Factor VII or VIIa Polypeptide Variants

ABSTRACT

The present invention relates to novel polypeptide variants of factor VII (FVII) or factor VIIa (FVIIa) polypeptides, where said variants comprise an amino acid substitution in position 10 and 32 and where said variants further comprise a sugar moiety covalently attached to an introduced in vivo N-glycosylation site located outside of the Gla domain. Such polypeptide variants are useful in therapy, in particular for the treatment of a variety of coagulation-related disorders, such as trauma.

FIELD OF THE INVENTION

The present invention relates to novel polypeptide variants of factorVII (FVII) or factor VIIa (FVIIa) polypeptides, where said variantscomprise an amino acid substitution in position 10 and 32 and where saidvariants further comprise a sugar moiety covalently attached to anintroduced in vivo N-glycosylation site.

The present invention also relates to use of such polypeptide variantsin therapy, in particular for the treatment of a variety ofcoagulation-related disorders.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually results in afibrin clot. Generally, the blood components participating in what hasbeen referred to as the “coagulation cascade” are proenzymes orzymogens, i.e. enzymatically inactive proteins that are converted intoan active form by the action of an activator. One of these coagulationfactors is FVII.

FVII is a vitamin K-dependent plasma protein synthesized in the liverand secreted into the blood as a single-chain glycoprotein with amolecular weight of 53 kDa (Broze & Majerus, J. Biol. Chem 1980;255:1242-1247). The FVII zymogen is converted into an activated form(FVIIa) by proteolytic cleavage at a single site, R152-I153, resultingin two chains linked by a single disulfide bridge. FVIIa in complex withtissue factor (FVIIa complex) is able to convert both factor IX andfactor X into their activated forms, followed by reactions leading torapid thrombin production and fibrin formation (Østerud & Rapaport, ProcNatl Acad Sci USA 1977; 74:5260-5264).

FVII undergoes post-translational modifications, including vitaminK-dependent carboxylation resulting in ten γ-carboxyglutamic acidresidues in the N-terminal region of the molecule. Thus, residue number6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO:2 areγ-carboxyglutamic acids residues in the Gla domain important for FVIIactivity. Other post-translational modifications include sugar moietyattachment at two naturally occurring N-glycosylation sites at position145 and 322, respectively, and at two naturally occurringO-glycosylation sites at position 52 and 60, respectively.

The gene coding for human FVII (hFVII) has been mapped to chromosome 13at q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). Itcontains nine exons and spans 12.8 Kb (O'Hara et al., Proc Natl Acad SciUSA 1987; 84:5158-5162). The gene organisation and protein structure ofFVII are similar to those of other vitamin K-dependent procoagulantproteins, with exons 1a and 1b encoding for signal sequence; exon 2 thepropeptide and Gla domain; exon 3 a short hydrophobic region; exons 4and 5 the epidermal growth factor-like domains; and exon 6 through 8 theserine protease catalytic domain (Yoshitake et al., Biochemistry 1985;24: 3736-3750).

Reports exist on experimental three-dimensional structures of hFVIIa(Pike et al., PNAS. U.S.A., 1999; 96:8925-30 and Kemball-Cook et al., J.Struct. Biol, 1999; 127:213-223); of hFVIIa in complex with solubletissue factor using X-ray crystallographic methods (Banner et al.,Nature, 1996; 380:41 and Zhang et al., J. Mol. Biol, 1999; 285: 2089);and of smaller fragments of hFVII (Muranyi et al., Biochemistry, 1998;37:10605 and Kao et al., Bio-chemistry, 1999; 38:7097).

Some protein-engineered variants of FVII have been reported. See, e.g.,Dickinson & Ruf, J Bio Chem, 1997; 272:19875-19879, Kemball-Cook et al.,J Biol Chem, 1998; 273:8516-8521, Bharadwaj et al., J Biol Chem, 1996;271:30685-30691, Ruf et al., Biochemistry, 1999; 38:1957-1966; WO99/20767; WO 00/11416; WO 02/22776; WO 02/38162; WO 01/83725; WO01/58935; U.S. Pat. No. 5,580,560.

Reports exist on expression of FVII in BHK or other mammalian cells (WO92/15686, WO 91/11514 and WO 88/10295) and co-expression of FVII andkex2 endoprotease in eukaryotic cells (WO 00/28065).

Commercial preparations of human recombinant FVIIa are sold asNovoSeven®. NovoSeven® is indicated for the treatment of bleedingepisodes in hemophilia A or B patients. NovoSeven® is the only rhFVIIafor effective and reliable treatment of bleeding episodes available onthe market.

An inactive form of FVII in which arginine 152 and/or isoleucine 153is/are modified has been reported in WO91/1154. These amino acids arelocated at the activation site. WO 96/12800 describes inactivation ofFVIIa by a serine proteinase inhibitor; inactivation by carbamylation ofFVIIa at the α-amino acid group I153 has been described by Petersen etal., Eur J Biochem, 1999; 261:124-129. The inactivated form is capableof competing with wild-type FVII or FVIIa for binding to tissue factorand inhibiting clotting activity. The inactivated form of FVIIa issuggested to be used for treatment of patients being in hypercoagulablestates, such as patients with sepsis, in risk of myocardial infarctionor of thrombotic stroke.

A circulating rhFVIIa half-life of 2.3 hours was reported in “SummaryBasis for Approval for NovoSeven®”, FDA reference number 96-0597.Relatively high doses and frequent administration are necessary to reachand sustain the desired therapeutic or prophylactic effect. As aconsequence adequate dose regulation is difficult to obtain and the needof frequent intravenous administrations imposes restrictions on thepatient's way of living.

In connection with treatment of uncontrolled bleedings, such as trauma,it is believed that factor VIIa is capable of activating factor X tofactor Xa without binding to tissue factor, and this activation reactionis believed to occur primarily on activated blood platelets (Hedner etal. Blood Coagulation & Fibrinolysis, 2000; 11; 107-111). However,hFVIIa or rhFVIIa has a low activity towards factor X in the absence oftissue factor and, consequently, treatment of uncontrolled bleeding, forexample in trauma patients, requires relatively high and multiple dosesof hFVIIa or rhFVIIa. Therefore, in order to treat uncontrolledbleedings more efficiently (to minimize blood loss) there is need forimproved FVIIa molecules, which possess a high activity toward factor Xin the absence of tissue factor. Such improved FVIIa molecules willexhibit a lowered clotting time (or faster action) as compared torhFVIIa when administered in connection with uncontrolled bleedings.

A molecule with a longer circulation half-life would decrease the numberof necessary administrations. Given the association of current therhFVIIa product with frequent injections, and the potential forobtaining more optimal therapeutic FVIIa levels with concomitantenhanced therapeutic effect, there is a clear need for improved FVII- orFVIIa-like molecules.

One way to increase the circulation half-life of a protein is to ensurethat renal clearance of the protein is reduced. This may be achieved byconjugating the protein to a chemical moiety, which is capable ofconferring reduced renal clearance to the protein.

Furthermore, attachment of a chemical moiety to the protein orsubstitution of amino acids exposed to proteolysis may effectively blocka proteolytic enzyme from contact leading to proteolytic degradation ofthe protein. Polyethylene glycol (PEG) is one such chemical moiety thathas been used in the preparation of therapeutic protein products. WO98/32466 suggests that FVII, among many other proteins, may be PEGylatedbut does not contain any further information in this respect. WO01/58935 discloses a new strategy for developing FVII or FVIIa moleculeshaving inter alia an increased half-life.

As indicated above, another problem in current rhFVIIa treatment is therelative instability of the molecule with respect to proteolyticdegradation. Proteolytic degradation is a major obstacle for obtaining apreparation in solution as opposed to a lyophilized product. Theadvantage of obtaining a stable soluble preparation lies in easierhandling for the patient, and, in the case of emergencies, quickeraction, which potentially can become life saving. Attempts to preventproteolytic degradation by site directed mutagenesis at majorproteolytic sites have been disclosed in WO 88/10295.

One object of the present invention is to provide improved FVII or FVIIamolecules (FVII or FVIIa variants) with a longer circulation half-life(thereby decreasing the number of necessary administrations) and whichare capable of activating factor X to factor Xa (without binding totissue factor) more efficiently than hFVIIa or rhFVIIa (thereby beingable to treat uncontrolled bleadings, such as a trauma, moreefficiently).

Another object of the present invention is to provide improved FVII orFVII molecules (FVII or FVIIa variants) with an increasedbioavailability (such as an increased Area Under the Curve as comparedto rhFVIIa when administered intravenously) and which are capable ofactivating factor X to factor Xa (without binding to tissue factor) moreefficiently than hFVIIa or rhFVIIa (thereby being able to treatuncontrolled bleadings, such as a trauma, more efficiently).

These objects are met by the FVII or FVIIa variants provided herein.

BRIEF DISCLOSURE OF THE INVENTION

In its broadest aspect the present invention relates to a FVII or FVIIapolypeptide variant having an amino acid sequence comprising 3-15 aminoacid modifications relative to hFVII or hFVIIa having the amino acidsequence shown in SEQ ID NO:2, wherein said amino acid sequence of thevariant comprises an amino acid substitution in position 10 and 32 andwherein a sugar moiety is covalently attached to an introduced in vivoN-glycosylation site located outside the Gla domain.

Another aspect of the invention relates to a nucleotide sequenceencoding the polypeptide variant of the invention.

In a further aspect the invention relates to an expression vectorcomprising the nucleotide sequence of the invention.

In a still further aspect the invention relates to a host cellcomprising the nucleotide sequence of the invention or the expressionvector of the invention.

In an even further aspect the invention relates to a pharmaceuticalcomposition comprising the polypeptide variant of the invention, and apharmaceutical acceptable carrier or excipient.

Still another aspect of the invention relates to the polypeptide variantof the invention, or the pharmaceutical composition of the invention,for use as a medicament.

Further aspects of the present invention will be apparent from the belowdescription as well as from the appended claims.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “conjugate” (or interchangeably “conjugated polypeptide”) isintended to indicate a heterogeneous (in the sense of composite orchimeric) molecule formed by the covalent attachment of one or morepolypeptide(s) to one or more non-polypeptide moieties such as polymermolecules, lipophilic compounds, sugar moieties or organic derivatizingagents. Preferably, the conjugate is soluble at relevant concentrationsand conditions, i.e. soluble in physiological fluids such as blood.Examples of conjugated polypeptides of the invention includeglycosylated and/or PEGylated polypeptides.

The term “covalent attachment” or “covalently attached” means that thepolypeptide variant and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through an intervening moiety or moieties, such asa bridge, spacer, or linkage moiety or moieties.

The term “non-polypeptide moiety” is intended to mean a molecule,different from a peptide polymer composed of amino acid monomers andlinked together by peptide bonds, which molecule is capable ofconjugating to an attachment group of the polypeptide variant of theinvention. Preferred examples of such molecules include polymermolecules, sugar moieties, lipophilic compounds or organic derivatizingagents. When used in the context of a conjugated variant of theinvention it will be understood that the non-polypeptide moiety islinked to the polypeptide part of the conjugated variant through anattachment group of the polypeptide. As explained above, thenon-polypeptide moiety can be directly covalently joined to theattachment group or it can be indirectly covalently joined to theattachment group through an intervening moiety or moieties, such as abridge spacer or linker moiety or moieties.

A “polymer molecule” is a molecule formed by covalent linkage of two ormore monomers, wherein none of the monomers is an amino acid residue,except where the polymer is human albumin or another abundant plasmaprotein. The term “polymer” may be used interchangeably with the term“polymer molecule”. The term is also intended to cover carbohydratemolecules attached by in vitro glycosylation, i.e. a syntheticglycosylation performed in vitro normally involving covalently linking acarbohydrate molecule to an attachment group of the polypeptide variant,optionally using a cross-linking agent. In vitro glycosylation isdiscussed in detail further below.

The term “sugar moiety” is intended to indicate acarbohydrate-containing molecule comprising one or more monosaccharideresidues, capable of being attached to the polypeptide variant (toproduce a polypeptide variant conjugate in the form of a glycosylatedpolypeptide variant) by way of in vivo glycosylation. The term “in vivoglycosylation” is intended to mean any attachment of a sugar moietyoccurring in vivo, i.e. during posttranslational processing in aglycosylating cell used for expression of the polypeptide variant, e.g.by way of N-linked and O-linked glycosylation. The exact oligosaccharidestructure depends, to a large extent, on the glycosylating organism inquestion.

An “N-glycosylation site” has the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine. Preferably, the amino acid residue in position +3relative to the asparagines residue is not a proline residue.

An “O-glycosylation site” is the OH-group of a serine or threonineresidue.

The term “attachment group” is intended to indicate a functional groupof the polypeptide variant, in particular of an amino acid residuethereof or a carbohydrate moiety, capable of attaching a non-polypeptidemoiety such as a polymer molecule, a lipophilic molecule, a sugar moietyor an organic derivatizing agent. Useful attachment groups and theirmatching non-polypeptide moieties are apparent from the table below.Conjugation Attachment Examples of non- method/-Activated group Aminoacid polypeptide moiety PEG Reference —NH₂ N-terminal, Polymer, e.g.PEG, mPEG-SPA Shearwater Inc. Lys with amide or imine Tresylated mPEGDelgado et al, critical group reviews in Therapeutic Drug CarrierSystems 9(3, 4): 249-304 (1992) —COOH C-terminal, Polymer, e.g. PEG,mPEG-Hz Shearwater Inc. Asp, Glu with ester or amide group CarbohydrateIn vitro coupling moiety —SH Cys Polymer, e.g. PEG, PEG-vinylsulphoneShearwater Inc. with disulfide, PEG-maleimide Delgado et al, criticalmaleimide or vinylsulfone reviews in group Therapeutic Drug CarbohydrateIn vitro coupling Carrier Systems moiety 9(3, 4): 249-304 (1992) —OHSer, Thr, Sugar moiety In vivo O-linked Lys, OH— PEG with ester,glycosylation ether, carbamate, carbonate —CONH₂ Asn as part Sugarmoiety In vivo N- of an N- Polymer, e.g. PEG glycosylation glycosylationsite Aromatic Phe, Tyr, Carbohydrate In vitro coupling residue Trpmoiety —CONH₂ Gln Carbohydrate In vitro coupling Yan and Wold, moietyBiochemistry, 1984, Jul 31; 23(16): 3759-65 Aldehyde Oxidized Polymer,e.g. PEG, PEGylation Andresz et al., 1978, Ketone oligosaccharidePEG-hydrazide Makromol. Chem. 179: 301, WO 92/16555, WO 00/23114Guanidino Arg Carbohydrate In vitro coupling Lundblad and Noyes, moietyChimical Reagents for Protein Modification, CRC Press Inc., Florida, USAImidazole His Carbohydrate In vitro coupling As for guanidine ringmoiety

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting aN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.

Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by in vivo N-glycosylation, the term“amino acid residue comprising an attachment group for a non-polypeptidemoiety” as used in connection with alterations of the amino acidsequence of the polypeptide is to be understood as meaning that one ormore amino acid residues constituting an in vivo N-glycosylation siteare to be altered in such a manner that a functional in vivoN-glycosylation site is introduced into the amino acid sequence.

In the present application, amino acid names and atom names (e.g. CA,CB, CD, CG, SG, NZ, N, O, C, etc) are used as defined by the ProteinDataBank (PDB) (www.pdb.org) based on the IUPAC nomenclature (IUPACNomenclature and Symbolism for Amino Acids and Peptides (residue names,atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984) together withtheir corrections in Eur. J. Biochem., 152, 1 (1985)).

The term “amino acid residue” is intended to indicate an amino acidresidue contained in the group consisting of alanine (Ala or A),cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E),phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H),isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine(Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln orQ), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine(Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

The terminology used for identifying amino acid positions is illustratedas follows: I205 indicates that position 205 is occupied by anisoleucine residue in the amino acid sequence shown in SEQ ID NO:2.I205T indicates that the isoleucine residue of position 205 has beensubstituted with a threonine residue. Alternative substitutions areindicated with a “/”, e.g. I205S/T means an amino acid sequence in whichisoleucine in position 205 is substituted with either serine orthreonine. Multiple substitutions are indicated with a “+”, e.g.K143N+N145T means a substitution of the lysine residue in position 143with an asparagine residue and a substitution of the asparagine residuein position 145 with a threonine residue. Insertion of an additionalamino acid residue is indicated in the following way: Insertion of atyrosine residue after A3 (i.e. in position 4) is indicated by A3AY(leading to insertion of a tyrosine residue in position 4). A deletionof an amino acid residue is indicated by an asterix. For example,deletion of a valine residue in position 172 is indicated by V172*.Simultaneous insertion and substitution are indicated in the followingway: Substitution of an alanine residue in position 175 with a threonineresidue followed by insertion of a leucine residue after position 175 isindicated A175TL.

Unless otherwise indicated, the numbering of amino acid residues madeherein is made relative to the amino acid sequence of the hFVII/hFVIIapolypeptide (SEQ ID NO:2).

The term “differs from” as used in connection with specific mutations isintended to allow for additional differences being present apart fromthe specified amino acid difference. For instance, in addition to theintroduction of in vivo N-glycosylation sites (located outside the Gladomain), the polypeptide may comprise other modifications that are notrelated to introduction of such amino acid residues. In a similar way,in addition to the modifications performed in the Gla domain aiming atincreasing the phospholipid membrane binding affinity, the polypeptidemay contain other modifications that are not necessarily related to thiseffect. Thus, in addition to the amino acid modifications disclosedherein, it will be understood that the amino acid sequence of thepolypeptide variant of the invention may, if desired, contain otheralterations, i.e. other substitutions, insertions or deletions. Thesemay, for example, include truncation of the N- and/or C-terminus by oneor more amino acid residues (e.g. by 1-10 amino acid residues), oraddition of one or more extra residues at the N- and/or C-terminus, e.g.addition of a methionine residue at the N-terminus or introduction of acysteine residue near or at the C-terminus, as well as “conservativeamino acid substitutions”, i.e. substitutions performed within groups ofamino acids with similar characteristics, e.g. small amino acids, acidicamino acids, polar amino acids, basic amino acids, hydrophobic aminoacids and aromatic amino acids.

Examples of such conservative substitutions are shown in the belowtable. 1 Alanine (A) Glycine (G) Serine (S) Threonine (T) 2 Asparticacid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)Histidine (H) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M)Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Still other examples of additional modifications are disclosed in thesection entitled “Other modifications outside the Gla domain” below.

The term “nucleotide sequence” is intended to indicate a consecutivestretch of two or more nucleotide molecules. The nucleotide sequence maybe of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “polymerase chain reaction” or “PCR” generally refers to amethod for amplification of a desired nucleotide sequence in vitro, asdescribed, for example, in U.S. Pat. No. 4,683,195. In general, the PCRmethod involves repeated cycles of primer extension synthesis, usingoligonucleotide primers capable of hybridising preferentially to atemplate nucleic acid.

The term “vector” refers to a plasmid or other nucleotide sequences thatare capable of replicating within a host cell or being integrated intothe host cell genome, and as such, are useful for performing differentfunctions in conjunction with compatible host cells (a vector-hostsystem): to facilitate the cloning of the nucleotide sequence, i.e. toproduce usable quantities of the sequence, to direct the expression ofthe gene product encoded by the sequence and to integrate the nucleotidesequence into the genome of the host cell. The vector will containdifferent components depending upon the function it is to perform.

“Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.

“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

“Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence coding for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide: apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that thenucleotide sequences being linked are contiguous and, in the case of asecretory leader, contiguous and in reading phase. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, then synthetic oligonucleotide adaptors or linkers areused, in conjunction with standard recombinant DNA methods.

In the context of the present invention the terms “modification” or“amino acid modification” is intended to cover replacement of an aminoacid side chain, substitution of an amino acid residue, deletion of anamino acid residue and/or insertion of an amino acid residue.

The terms “mutation” and “substitution” are used interchangeably herein.

The term “introduce” refers to introduction of an amino acid residue bysubstitution of an existing amino acid residue or, alternatively, byinsertion of an additional amino acid residue.

The term “remove” refers to removal of an amino acid residue bysubstitution of the amino acid residue to be removed by another aminoacid residue or, alternatively, by deletion (without substitution) ofthe amino acid residue to be removed

The term “FVII” or “FVII polypeptide” refers to a FVII molecule providedin single chain form. One example of a FVII polypeptide is wild-typehuman FVII (hFVII) shown in SEQ ID NO:2. It should be understood,however, that the term “FVII polypeptide” also covers hFVII-likemolecules, such as fragments or variants of SEQ ID NO:2, in particularvariants where the sequence comprises at least one, such as 1-15, e.g.,1-10, amino acid modifications as compared to SEQ ID NO:2.

The term “FVIIa” or “FVIIa polypeptide” refers to a FVIIa moleculeprovided in its activated two-chain form. When the amino acid sequenceof SEQ ID NO:2 is used to describe the amino acid sequence of FVIIa itwill be understood that the peptide bond between R152 and I153 of thesingle-chain form has been cleaved, and that one of the chains comprisesamino acid residues 1-152, the other chain amino acid residues 153-406.

The terms “rFVII” and “rFVIIa” refer to FVII and FVIIa polypeptidesproduced by recombinant techniques.

The terms “hFVII” and “hFVIIa” refer to human wild-type FVII and FVIIa,respectively, having the amino acid sequence shown in SEQ ID NO:2

The terms “rhFVII” and “rhFVIIa” refer to human wild-type FVII andFVIIa, having the amino acid sequence shown in SEQ ID NO:2, produced byrecombinant means. An example of rhFVIIa is NovoSeven®.

When used herein, the term “Gla domain” is intended to cover amino acidresidues no. 1 to 45 of SEQ ID NO:2.

Accordingly, the term “located outside the Gla domain” covers amino acidresidue no. 46-406 of SEQ ID NO:2.

The abbreviations “TF” and “TFPI” mean Tissue Factor and Tissue FactorPathway Inhibitor, respectively.

The term “protease domain” is used about residues 153-406 counted fromthe N-terminus.

The term “catalytic site” is used to mean the catalytic triad consistingof S344, D242 and H193 of the polypeptide variant.

The term “parent” is intended to indicate the molecule to bemodified/improved in accordance with the present invention. Although theparent polypeptide to be modified by the present invention may be anyFVII or FVIIa polypeptide, and thus be derived from any origin, e.g. anon-human mammalian origin, it is preferred that the parent polypeptideis hFVII or hFVIIa

A “variant” is a polypeptide, which differs in one or more amino acidresidues from its parent polypeptide, normally in 3-15 amino acidresidues (e.g. in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 aminoacid residues), such as in 3-10 amino acid residues, e.g. in 3-8 or 3-5amino acid residues. In other words, a “variant” typically contains 3-15amino acid modifications (for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 amino acid modifications), such as 3-10 amino acidmodifications, e.g. 3-8 or 3-5 amino acid modifications relative to theparent polypeptide. In the present context, the term “modification”encompasses insertions, deletions, substitutions and combinationsthereof. It will be understood that a polypeptide variant according tothe present invention will be modified in at least three positions,namely in at least position 10 and 32 (located in the Gla domain) and inat least one position located outside the Gla domain, where said atleast one modification creates an in vivo N-glycosylation site.

The term “clotting activity” is used to mean the activity measured inthe “Clotting Assay” described herein. In order to exhibit “clottingactivity” a variant of the invention, in its activated form, should haveat least 10% of the clotting acitivty of rhFVIIa when assayed in the“Clotting Assay” described herein. In a preferred embodiment of theinvention the variant, in its activated form, has at least 20% of theclotting activity of rhFVIIa, such as at least 30%, e.g. at least 40%,more preferably at least 50%, such as at least 60%, e.g. at least 70%,even more preferably at least 80%, such as at least 90% of the clottingactivity of rhFVIIa when assayed in the “Clotting Assay” describedherein. In an interesting embodiment the variant, in its activated form,has substantially the same clotting activity as rhFVIIs, such as aclotting activity of 75-125% of the clotting acitivity of rhFVIIa.

The term “amidolytic activity” is used to mean the activity measured inthe “Amidolytic Assay” described herein. In order to exhibit “amidolyticactivity” a variant of the invention, in its activated form, should haveat least 10% of the amidolytic acitivty of rhFVIIa when assayed in the“Amidolytic Assay” described herein. In a preferred embodiment of theinvention the variant, in its activated form, has at least 20% of theamidolytic activity of rhFVIIa, such as at least 30%, e.g. at least 40%,more preferably at least 50%, such as at least 60%, e.g. at least 70%,even more preferably at least 80%, such as at least 90% of theamidolytic activity of rhFVIIa when assayed in the “Amidolytic Assay”described herein. In an interesting embodiment the variant, in itsactivated form, has substantially the same amidolytic activity asrhFVIIs, such as an amidolytic activity of 75-125% of the amidolyticacitivity of rhFVIIa.

In the present context, the term “activity” is also used in connectionwith the variants capability of activating FX to FXa. This activity isalso denoted “FX activation activity” or “FXa generation activity”.

The term “increased FX activiation activity” or “increased FXageneration activity” is used to indicate that a variant of theinvention, in its activated form, has a statistically signifycantlyincreased capability to activate FX to FXa as compared to rhFVIIa or. Towhat extent a variant of the invention (in its activated form) has anincreased FX activation activity may conveniently be determined in the“TF-independent Factor X Activation Assay” described herein.

The term “stronger clot” or “increased clot strength” is used toindicate that the strength of the clot generated by the polypeptidevariant is statistically significantly increased relative to thatgenerated by rhFVIIa as determined under comparable conditions. Thiseffect may be determined as the Area Under the Curve (AUC_(throm))generated by the variant of the invention, in its activated form, whenassayed in the “Thrombogram Assay” disclosed herein. In a similar way,the term “increased AUC_(throm)” is used to indicate that the Area Underthe Curve generated by the variant (in its activated form) isstatistically significantly increased relative to that generated byrhFVIIa as determined under comparable conditions and when measured inthe “Thrombogram Assay” described herein

The term “T_(max)” is used about the time it takes to obtain the maximumthrombin activity level in the “Thrombogram Assay”.

The term “immunogenicity” as used in connection with a given substanceis intended to indicate the ability of the substance to induce aresponse from the immune system. The immune response may be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology(8^(th) Edition, Blackwell) for further definition of immunogenicity).Normally, reduced antibody reactivity will be an indication of reducedimmunogenicity. The reduced immunogenicity may be determined by use ofany suitable method known in the art, e.g. in vivo or in vitro.

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value.

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternatively terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The polypeptide is cleared by theaction of one or more of the reticuloendothelial systems (RES), kidney,spleen or liver, by tissue factor, SEC receptor or other receptormediated elimination, or by specific or unspecific proteolysis.Normally, clearance depends on size (relative to the cutoff forglomerular filtration), charge, attached carbohydrate chains, and thepresence of cellular receptors for the protein. The functionality to beretained is normally selected from procoagulant, proteolytic or receptorbinding activity. The functional in vivo half-life and the serumhalf-life may be determined by any suitable method known in the art.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of thepolypeptide variant is statistically significantly increased relative tothat of rhFVIIa as determined under comparable conditions (typicallydetermined in an experimental animal, such as rats, rabbits, pigs ormonkeys).

The term “AUC_(iv)” or “Area Under the Curve when administeredintravenously” is used in its normal meaning, i.e. as the area under theactivity in serum-time curve, where the polypeptide variant has beenadministered intravenously, in particular when administeredintravenously in rats. Typically, the activity measured is the “clottingactivity” as defined hereinbefore. Once the experimental activity-timepoints have been determined, the AUC_(iv) may conveniently be calculatedby a computer program, such as GraphPad Prism 3.01.

It will be understood that in order to make a direct comparison betweenthe AUC_(iv)-values of different molecules (e.g. between the variants ofthe invention and a reference molecule such as rhFVIIa) the same amountof activity should be administered. Consequently, the AUC_(iv)-valuesare typically normalized (i.e. corrected for differences in the injecteddose) and expressed as AUC_(iv)/dose administered.

The term “reduced sensitivity to proteolytic degradation” is primarilyintended to mean that the polypeptide variant has reduced sensitivity toproteolytic degradation in comparison to hFVIIa or rhFVIIa as determinedunder comparable conditions. Preferably, the proteolytic degradation isreduced by at least 10% (e.g. by 10-25% or by 10-50%), such as at least25% (e.g. by 25-50%, by 25-75% or by 25-100%), more preferably by atleast 35%, such as at least 50%, (e.g. by 50-75% or by 50-100%) evenmore preferably by at least 60%, such as by at least 75% (e.g. by75-100%) or even at least 90%. Most preferably, the proteolyticdegradation is reduced by at least 100%.

The term “renal clearance” is used in its normal meaning to indicate anyclearance taking place by the kidneys, e.g. by glomerular filtration,tubular excretion or degradation in the tubular cells. Renal clearancedepends on physical characteristics of the polypeptide, including size(diameter), hydrodynamic volume, symmetry, shape/rigidity, and charge.Normally, a molecular weight of about 67 kDa is considered to be acut-off-value for renal clearance. Renal clearance may be established byany suitable assay, e.g. an established in vivo assay. Typically, renalclearance is determined by administering a labelled (e.g. radiolabelledor fluorescence labelled) polypeptide to a patient and measuring thelabel activity in urine collected from the patient. Reduced renalclearance is determined relative to a corresponding referencepolypeptide, e.g. rhFVIIa, under comparable conditions. Preferably, therenal clearance rate of the polypeptide variant is reduced by at least50%, preferably by at least 75%, and most preferably by at least 90%compared to rhFVIIa.

The terms “at least 25% of its side chain exposed to the surface of themolecule” and “at least 50% of its side chain exposed to the surface ofthe molecule” are defined with reference to Example 1, where thecalculations, etc. are described in detail.

It should be noted that when the terms “at least 25% of its side chainexposed to the surface of the molecule” and “at least 50% of its sidechain exposed to the surface of the molecule” are used in connectionwith introduction of an in vivo N-glycosylation site these terms referto the surface accessibility of the amino acid side chain in theposition where the sugar moiety is actually attached. In many cases itwill be necessary to introduce a serine or a threonine residue inposition +2 relative to the asparagine residue to which the sugar moietyis actually attached (unless, of course, this position is alreadyoccupied by a serine or a threonine residue) and these positions, wherethe serine or threonine residues are introduced, are allowed to beburied, i.e. to have less than 25% or 50% of their side chains exposedto the surface of the molecule.

The terms “tissue factor binding site”, “active site region” and “ridgeof the active site binding cleft” are defined with reference to Example1 herein, wherein the above-mentioned sites/regions are determined.

The term “hydrophobic amino acid residue” includes the following aminoacid residues: isoleucine (I), leucine (L), methionine (M), valine (V),phenylalanine (F), tyrosine (Y) and tryptophan (W).

The term “negatively charged amino acid residue” includes the followingamino acid residues: Aspartic acid (D) and glutamic acid (E).

The term “positively charged amino acid residue” includes the followingamino acid residues: Lysine (K), arginine (R) and histidine (H).

Variants of the Invention

In its broadest aspect the present invention relates to a FVII or FVIIapolypeptide variant having an amino acid sequence comprising 3-15 aminoacid modifications relative to hFVII or hFVIIa having the amino acidsequence shown in SEQ ID NO:2, wherein said amino acid sequence of thevariant comprises an amino acid substitution in position 10 and 32 andwherein a sugar moiety is covalently attached to an introduced in vivoN-glycosylation site located outside the Gla domain.

The modifications perfomed in the two above-indicated regions of theparent FVII polypeptide serve the following purposes:

The modifications performed in positions located outside the Gla domain(introduction of in vivo N-glycosylation site(s)) are preferably of suchnature that the AUC_(iv), the functional in vivo half-life and/or theserum half-life of the resulting variant is increased as compared torhFVIIa.

The modifications perfomed in the Gla domain of the parent polypeptideare preferably of such nature that an increased phospholipid membranebinding affinity of the resulting molecule is achieved and/or of suchnature the resulting molecule has an improved capability to activate FXto FXa and/or of such nature that a stronger clot is formed.

Without being limited by any particular theory, it is presently believedthat the enhanced membrane affinity results in a higher localconcentration of the activated polypeptide variants in close proximityto the other coagulation factors, particularly FX. Thus, the rate ofactivation of FX to FXa will be higher, simply due to a higher molarratio of the activated FVII variant to FX. The increased activation rateof FX then results in a higher amount of active thrombin, and thus ahigher rate of cross-linking of fibrin.

Thus, in preferred embodiments of the invention the parent FVII or FVIIapolypeptide has been modified so that the resulting activatedpolypeptide variant has (as compared to rhFVIIa):

-   -   i) an increased bioavailability (AUC_(iv)) and an increased        phospholipid membrane binding affinity;    -   ii) an increased bioavailability (AUC_(iv)) and an increased        capability to activate FX to FXa;    -   iii) an increased bioavailability (AUC_(iv)) and is capable of        generating a stronger clot (increased AUC_(throm));    -   iv) an increased bioavailability (AUC_(iv)) and a reduced        T_(max);    -   v) an increased functional in vivo half-life and an increased        phospholipid membrane binding affinity;    -   vi) an increased functional in vivo half-life and an increased        capability to activate FX to FXa;    -   vii) an increased functional in vivo half-life and is capable of        generating a stronger clot (increased AUC_(throm));    -   viii) an increased functional in vivo half-life and a reduced        T_(max);    -   ix) an increased serum half-life and an increased phospholipid        membrane binding affinity;    -   x) an increased serun half-life and an increased capability to        activate FX to FXa;    -   xi) an increased serum half-life and is capable of generating a        stronger clot (increased AUC_(throm)); and/or    -   xii) an increased serum half-life and a reduced T_(max).

Consequently, medical treatment with a polypeptide variant according tothe invention offers a number of advantages over the currently availablerhFVIIa compound (NovoSeven®), such as administration of lower dosage,longer duration between injections, increased clot strength and/orfaster action.

Thus, preferred variants of the invention are such variants which, intheir activated forms and when compared to rhFVIIa, generates inincreased Area Under the Curve when administered intravenously(AUC_(iv)), in particular when administered intravenously in rats. Moreparticularly, variants of the present invention, which are preferred,are such variants where the ratio between the AUC_(iv) of said variant,in its actvated form, and the AUC_(iv) of rhFVIIa is at least 1.25, suchas at least 1.5, e.g. at least 1.75, more preferably at least 2, such asat least 3, even more preferably at least 4, such as at least 5, inparticular when administered (intravenously) in rats.

This effect may in turn (but do not necessarily do so) correspond to anincreased functional in vivo half-life and/or an increased serumhalf-life as compared to rhFVIIa. Accordingly, in another preferredembodiment of the invention, the ratio between the functional in vivohalf-life or the serum half-life for the variant, in its activated form,and the functional in vivo half-life or the serum half-life for rhFVIIais at least 1.25. More preferably, the ratio between the relevanthalf-life for the variant, in its activated form, and the relevanthalf-life for hFVIIa or rhFVIIa is at least 1.5, such as at least 1.75,e.g. at least 2, even more preferably at least 3, such as at least 4,e.g. at least 5.

As will be understood, the variants of the invention also possess, inaddition to the above-mentioned functionality (i.e. increased AUC_(iv),increased functional in vivo half-life and/or increased serumhalf-life), an increased phospholipid membrane binding affinity ascompared to rhFVII, an an increased capability to activate FX to Fxa, acapability of generating a stronger clot (increased AUC_(throm)) and/ora reduced T_(max).

Thus, in one preferred embodiment of the invention, the polypeptidevariant has (in addition to an increased AUC_(iv), increased functionalin vivo half-life and/or increased serum half-life) an increasedphospholipid membrane binding affinity relative to rhFVIIa. Membranebinding affinity may be measured by methods known in the art, such as bythe Biacore® assay described in K. Nagata and H. Handa (Ads.), Real-TimeAnalysis of Biomolecular Interactions, Springer-Verlag, Tokyo, 2000,Chapter 6 entitled “Lipid-Protein Interactions”. Alternatively, themembrane binding affinity may be measured as described in Example 1 inWO 99/20767.

In another preferred embodiment of the invention, the polypeptidevariant (in addition to an increased AUC_(iv), increased functional invivo half-life and/or increased serum half-life), has an increased FXactivation activity as compared to rhFVIIa, in particular when assayedin a TF-independent assay, such as the “TF-independent Factor XActivation Assay” disclosed herein. More particularly, it is preferredthat the ratio between the FX activation activity of the polypeptidevariant, in its activated form, and the FX activation activity ofrhFVIIa is at least 1.25 when assayed in the “TF-independent Factor XActivation Assay” disclosed herein. More preferably, the ratio betweenthe FX activation activity of the variant, in its activated form, andthe FX activation activity of rhFVIIa is at least 1.5, such as at least1.75, e.g. at least 2, even more preferably at least 3, such as at least4, e.g. at least 5, still more preferably at least 6, such as at least7, e.g. at least 8, most preferably at least 9, such as at least 10,when assayed in the “TF-independent Factor X Activation Assay” describedherein.

In still another preferred embodiment of the invention, the polypeptidevariant is (in addition to an increased AUC_(iv), increased functionalin vivo half-life and/or increased serum half-life), in its activatedform, capable of generating a stronger clot as compared to rhFVIIa. Thiseffect may be determined in the “Thrombogram Assay” described herein asan increase in the Area Under the Curve (AUC_(throm)). The AUC_(throm)is also sometimes denoted “total thrombin work” and constitutes ameasure for the strength of the clot formed. More particularly, it ispreferred that the ratio between the AUC_(throm), generated by thevariant in its activated form, and the AUC_(throm) generated by rhFVIIais at least 1.15 when assayed in the “Thrombogram Assay” describedherein. More preferably, the ratio is at least 1.2, such as at least1.25, e.g. at least 1.3, even more preferably at least 1.4, such as atleast 1.5, e.g. at least 1.6, most preferably at least 1.7, such as atleast 1.8, e.g. at least 1.9 or at least 2.

In even another preferred embodiment of the invention the polypeptidevariant has (in addition to an increased AUC_(iv), increased functionalin vivo half-life and/or increased serum half-life), in its activatedform, a faster action. This effect may be determined in the “ThrombogramAssay” described herein as a reduction in the time needed to reachmaximum thrombin level (T_(max)). Accordingly, preferred variants aresuch variants where the ratio between T_(max) for the variant, in itsactivated form, and T_(max) for rhFVIIa is at the most 0.95 when assayedin the “Thrombogram Assay” described herein. Preferably, the ratio is atthe most 0.9, such as at the most 0.8, e.g. at the most 0.7, morepreferably at the most 0.6, such as at the most 0.5.

Introduction of In Vivo N-glycosylation Sites Located Outside the GlaDomain

A number of suitable modifications leading to an increase in AUC_(iv),functional in vivo half-life and/or serum half-life is disclosed in WO01/58935. The variants disclosed in WO 01/58935 are the result of agenerally new strategy for developing improved FVII or FVIIa molecules,which may also be used for the parent FVII or FVIIa polypeptide of thepresent invention.

The position to be modified is preferably selected from a part of theFVII or FVIIa molecule that is located outside the tissue factor bindingsite, and/or outside the active site region, and/or outside the ridge ofthe active site binding cleft. These sites/regions are identified inExample 1 herein. It should be emphasized, however, that in certainsituations, e.g. in case an inactivated polypeptide variant is desired,it may be advantageous to perform modifications in or close to suchregions. For example, it is contemplated that one or more in vivoN-glycosylation sites may advantageously be introduced in the activesite region or at the ridge of the active site binding cleft of the FVIIor FVIIa molecule. The active site region, the tissue factor bindingsite and the ridge of the active site binding cleft are defined inExample 1 herein and are constituted by the following residues:

I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181,V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201,V228, I229, I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239,N240, H241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285,Q286, T293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342,D343, S344, G345, G346, P347, H348, L358, T359, G360, 1361, V362, S363,W364, G365, C368, V376, Y377, T378, R379, V380, Q382, Y383, W386, L387,L400 and F405 (active site region);

L13, K18, F31, E35, R36, L39, F40, I42, S43, S60, K62, D63, Q64, L65,I69, C70, F71, C72, L73, P74, F76, E77, G78, R79, E82, K85, Q88, I90,V92, N93, E94, R271, A274, F275, V276, R277, F278, R304, L305, M306,T307, Q308, D309, Q312, Q313, E325 and R379 (tissue factor bindingsite); and

N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370 (theridge of the active site binding cleft).

The total number of amino acid residues to be modified outside the Gladomain in the parent FVII or FVIIa polypeptide (as compared to the aminoacid sequence shown in SEQ ID NO:2) will typically not exceed 10.Preferably, the FVII or FVIIa variant comprises an amino acid sequencewhich differs in 1-10 amino acid residues from amino acid residues46-406 shown in SEQ ID NO:2, typically in 1-8 or in 2-8 amino acidresidues, e.g. in 1-5 or in 2-5 amino acid residues, such as in 1-4 orin 1-3 amino acid residues, e.g. in 1, 2 or 3 amino acid residues fromamino acid residues 46-406 shown in SEQ ID NO:2.

Thus, the polypeptide variant of the invention may contain 1-10(additional or introduced) in vivo N-glycosylation sites, typically 1-8or 2-8 (additional or introduced) in vivo N-glycosylation sites,preferably 1-5 or 2-5 (additional or introduced) in vivo N-glycosylationsites, such as 1-4 or 1-3 (additional or introduced) in vivoN-glycosylation sites, e.g. 1, 2 or 3 (additional or introduced) in vivoN-glycosylation sites. Analogously, the polypeptide variant of theinvention may contain 1-10 (additional or introduced) sugar moieties,typically 1-8 or 2-8 (additional or introduced) sugar moieties,preferably 1-5 or 2-5 (additional or introduced) sugar moieties, such as1-4 or 1-3 (additional or introduced) sugar moieties, e.g. 1, 2 or 3(additional or introduced) sugar moieites. It will be understood thatthe introduced sugar moiety/moieties will be covalently attached to theintroduced in vivo N-glycosylation site(s)

When used in the present context, the term “naturally occurringglycosylation site” covers the glycosylation sites at postions N145,N322, S52 and S60. In a similar way, the term “naturally occurring invivo O-glycosylation site” includes the positions S52 and S60, whereasthe term “naturally occurring in vivo N-glycosylation site” includespositions N145 and N322.

It will be understood that in order to prepare a polypeptide variant,wherein the polypeptide variant comprises one or more sugar moietiescovalently attached to one or more in vivo N-glycosylation sites, thepolypeptide variant must be expressed in a host cell capable ofattaching sugar (oligosaccharide) moieties at the glycosylation site(s)or alternatively subjected to in vitro glycosylation. Examples ofglycosylating host cells are given in the section further below entitled“Coupling to a sugar moiety”.

Examples of positions, wherein the in vivo N-glycosylation sites may beintroduced include, but is not limited to, positions comprising an aminoacid residue having an amino acid residue having at least 25% of itsside chain exposed to the surface (as defined in Example 1 herein), suchas in a position comprising an amino acid residue having at least 50% ofits side chain exposed to the surface (as defined in Example 1 herein).In general, it is preferred that the in vivo N-glycosylation site isintroduced by substitution, although insertion is also contemplated. Theposition is preferably selected from a part of the molecule that islocated outside the tissue factor binding site and/or the active siteregion and/or outside the ridge of the active site cleft. Thesesites/regions are identified in Example 1 herein. It should beunderstood that when the term “at least 25% (or at least 50%) of itsside chain exposed to the surface” is used in connection withintroduction of an in vivo N-glycosylation site this term refers to thesurface accessibility of the amino acid side chain in the position wherethe sugar moiety is actually attached. In many cases it will benecessary to introduce a serine or a threonine residue in position +2relative to the asparagine residue to which the sugar moiety is actuallyattached (unless, of course, this position is already occupied by aserine or a threonine residue) and these positions, where the serine orthreonine residues are introduced, are allowed to be buried, i.e. tohave less than 25% of their side chains exposed to the surface.

Specific and preferred examples of such substitutions creating an invivo N-glycosylation site include a substitution selected from the groupconsisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T,I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T,R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N, D334N andcombinations thereof. More preferably, the in vivo N-glycosylation siteis introduced by a substitution selected from the group consisting ofA51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, I205T, V253N,T267N+S269T, S314N+K316T, R315N+V317T, K316N+G318T, G318N, D334N andcombinations thereof. Even more preferably, the in vivo N-glycosylationsite is introduced by a substitution selected from the group consistingof T106N, A175T, I205T, V253N, T267N+S269T and combinations thereof, inparticular I205T.

In one embodiment, only one in vivo N-glycosylation site has beenintroduced by substitution. In another embodiment, two or more (such astwo) in vivo N-glycosylation sites have been introduced by substitution.Examples of preferred substitutions creating two in vivo N-glycosylationsites include substitutions selected from the group consisting ofA51N+G58N, A51N+T106N, A51N+K109N, A51N+G124N, A51N+K143N+N145T,A51N+A175T, A51N+I205T, A51N+V253N, A51N+T267N+S269T, A51N+S314N+K316T,A51N+R315N+V317T, A51N+K316N+G318T, A51N+G318N, A51N+D334N, G58N+T106N,G58N+K109N, G58N+G124N, G58N+K143N+N145T, G58N+A175T, G58N+I205T,G58N+V253N, G58N+T267N+S269T, G58N+S314N+K316T, G58N+R315N+V317T,G58N+K316N+G318T, G58N+G318N, G58N+D334N, T106N+K109N, T106N+G124N,T106N+K143N+N145T, T106N+A175T, T106N+I205T, T106N+V253N,T106N+T267N+S269T, T106N+S314N+K316T, T106N+R315N+V317T,T106N+K316N+G318T, T106N+G318N, T106N+D334N, K109N+G124N,K109N+K143N+N145T, K109N+A175T, K109N+I205T, K109N+V253N,K109N+T267N+S269T, K109N+S314N+K316T, K109N+R315N+V317T,K109N+K316N+G318T, K109N+G318N, K109N+D334N, G124N+K143N+N145T,G124N+A175T, G124N+I205T, G124N+V253N, G124N+T267N+S269T,G124N+S314N+K316T, G124N+R315N+V317T, G124N+K316N+G318T, G124N+G318N,G124N+D334N, K143N+N145T+A175T, K143N+N145T+I205T, K143N+N145T+V253N,K143N+N145T+T267N+S269T, K143N+N145T+S314N+K316T,K143N+N145T+R315N+V317T, K143N+N145T+K316N+G318T, K143N+N145T+G318N,K143N+N145T+D334N, A175T+I205T, A175T+V253N, A175T+T267N+S269T,A175T+S314N+K316T, A175T+R315N+V317T, A175T+K316N+G318T, A175T+G318N,A175T+D334N, I205T+V253N, I205T+T267N+S269T, I205T+S314N+K316T,I205T+R315N+V317T, I205T+K316N+G318T, I205T+G318N, I205T+D334N,V253N+T267N+S269T, V253N+S314N+K316T, V253N+R315N+V317T,V253N+K316N+G318T, V253N+G318N, V253N+D334N, T267N+S269T+S314N+K316T,T267N+S269T+R315N+V317T, T267N+S269T+K316N+G318T, T267N+S269T+G318N,T267N+S269T+D334N, S314N+K316T+R315N+V317T, S314N+K316T+G318N,S314N+K316T+D334N, R315N+V317T+K316N+G318T, R315N+V317T+G318N,R315N+V317T+D334N and G318N+D334N. More preferably, the substitutionsare selected from the group consisiting of T106N+A175T, T106N+I205T,T106N+V253N, T106N+T267N+S269T, A175T+I205T, A175T+V253N,A175T+T267N+S269T, I205T+V253N, I205T+T267N+S269T and V253N+T267N+S269T,even more preferably from the group consisiting of T106N+I205T,T106N+V253N and I205T+T267N+S269T.

In an even further embodiment, three or more (such as three) in vivoN-glycosylation sites have been introduced by substitution. Examples ofpreferred substitutions creating three in vivo N-glycosylation sitesinclude substitutions selected from the group consisiting ofI205T+V253N+T267N+S269T and T106N+I205T+V253N.

As discussed above, it is preferred that the in vivo N-glycosylationsite is introduced in a position which does neither form part of thetissue factor binding site nor form part of the active site region andthe ridge of the active site binding cleft as defined herein. It isenvisaged that such glycosylation variants will primarily belong to theclass of active polypeptide variants as defined hereinbefore.

It will be understood that as an alternative to introduction of in vivoN-glycosylation sites in the above-discussed positions one may introduce(either by substitution or by insertion) cysteine residues in the samepositions, where the introduced cysteine residue is then subsequentlycovalently attached to a non-polypeptide moiety, such as PEG, inparticular mPEG. Thus, examples of positions, wherein a cysteine residuemay be introduced include, but is not limited to, positions comprisingan amino acid residue having an amino acid residue having at least 25%of its side chain exposed to the surface (as defined in Example 1herein), such as in a position comprising an amino acid residue havingat least 50% of its side chain exposed to the surface (as defined inExample 1 herein). The position is preferably selected from a part ofthe molecule that is located outside the tissue factor binding siteand/or the active site region and/or outside the ridge of the activesite cleft. These sites/regions are identified in Example 1 herein.Thus, the above disclosure concerning introduction of in vivoN-glycosylation sites applies mutatis mutandis to introduction ofcysteine residues.

It will be understood that the modifications in positions locatedoutside the Gla domain discussed in the above section should be combinedwith one or more modifications in the Gla domain (see the sectionentitled “Modifications in the Gla domain” below).

Modifications in the Gla Domain

As will be understood the variants of the present invention comprises,in addition to at least one introduced in vivo N-glycosylation sitelocated outside the Gla domain (cf. above), at least two substitutionsin the Gla domain, namely a substitution in position 10 and position 32.

A number of suitable modifications leading to increased phospholipidmembrane binding affinity is disclosed in WO 99/20767 and WO 00/66753.

In a preferred embodiment of the invention the substitution in position10 is P10Q. In another preferred embodiment of the invention thesubstitution in position 32 is K32E. In a particularly preferredembodiment of the invention the variant comprises the followingsubstitutions P10Q+K32E.

In an interesting embodiment of the invention the variant comprises, inaddition to the substitutions in position 10 and 32, such as in additionto the substitutions P10Q+K32E, at least one further modification in theGla domain.

In one preferred embodiment of the invention the further modification inthe Gla domain comprises an amino acid substitution in position 33.Preferably, a hydrophobic amino acid residue is introduced bysubstitution in position 33, such as D33I, D33L, D33M, D33V, D33F, D33Yor D33W, in particular D33F. Accordingly, in one very interestingembodiment of the invention, the variant comprises the followingsubstitutions P10Q+K32+D33F.

In another preferred embodiment of the invention the furthermodification in the Gla domain comprises an insertion of at least one(such as one) amino acid residue between position 3 and 4. It ispreferred that the inserted amino acid residue is a hydrophobic aminoacid residue. Most preferably the insertion is A3AY. Accordingly, inanother very interesting embodiment of the invention, the variantcomprises the following modifications A3AY+P10Q+K32E orA3AY+P10Q+K32E+D33F.

In still another preferred embodiment of the invention the furthermodification in the Gla domain comprises a substitution in position 34.It is preferred that a negatively charged amino acid residue isintroduced by substitution in position 34. Most preferably thesubstitution is A34E. Accordingly, in still another very interestingembodiment of the invention, the variant comprises the followingmodifications P10Q+K32E+A34E, P10Q+K32E+D33F+A34E, A3AY+P10Q+K32E+A34Eor A3AY+P10Q+K32E+D33F+A34E.

The Gla domain may also contain modifications in other positions, inparticular in positions 8, 11 and 28, such as R28F or R28E. On the otherhand it should be understood that the Gla domain should not be modifiedto such an extent that the membrane binding properties are impaired.Accordingly, it is preferred that no modifications are made in theresidues that become 7-carboxylated, i.e. it is preferred that nomodifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and35. In a similar way, it is in general not preferred thatnon-polypeptide moieties, such as sugar moieties and/or PEG groups, areintroduced in the Gla domain. Consequently, it is preferred that nomodifications are made in the Gla domain that creates an in vivoN-glycosylation site.

Finally, it will be understood that the modifications in the Gla domaindiscussed in this section must be combined with one or more of themodifications disclosed in the section entitled “Introduction of in vivoN-glycosylation sites located outside the Gla domain” above.

Specific examples of such “combined” variants are given below.

In one embodiment of the invention said FVII or FVIIa variant comprisesthe following modifications: P10Q+K32E+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+I205T+T267N+S269T. In afurther embodiment of the invention said FVII or FVIIa variant comprisesthe following modifications: A3AY+P10Q+K32E+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+V253N.

In a further preferred embodiment of the invention said FVII or FVIIavariant comprises the following modifications:A3AY+P10Q+K32E+T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+I205T+T267N+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+I205T+T267N+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+T205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications:A3AY+P10Q+K32E+D33F+I205T+T267N+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+A34E+I205T+T267N+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34E T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: P10Q+K32E+D33F+A34EI205T+T267N+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E T267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E T106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34E T106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+A34EI205T+T267N+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34E+T106N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34E A175T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34E I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34E V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34ET267+S269T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34ET106N+I205T.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34ET106N+V253N.

In a further embodiment of the invention said FVII or FVIIa variantcomprises the following modifications: A3AY+P10Q+K32E+D33F+A34EI205T+T267N+S269T.

Other Modifications Outside the Gla Domain

In a further embodiment of the present invention, the FVII or FVIIavariant may, in addition to the modifications described in the sectionsabove, also contain mutations, which are already known to increase theintrinsic activity of the polypeptide, e.g. such as those described inWO 02/22776

Examples of preferred substitutions include substitutions selected fromthe group consisting of V158D, E296D, M298Q, L305V and K337A. Morepreferably, said substitutions are selected from the group consisting ofV158D+E296D+M298Q+L305V+K337A, V158D+E296D+M298Q+K337A,V158D+E296D+M298Q+L305V, V158D+E296D+M298Q, M298Q, L305V+K337A, L305Vand K337A.

In a further embodiment of the present invention, the FVII or FVIIavariant may, in addition to the modifications described in the sectionsabove, also contain mutations, which are already known to cause adecreased inhibition by TFPI. One example includes the substitutionK341Q disclosed by Neuenschwander et al, Biochemistry, 1995;34:8701-8707.

Moreover, the variant may contain modifications which are belived toincrease the TF binding affinity. Examples of such modifications includesubstitutions selected from the group consisting of L39E, L39Q, L39H,I42K, I42R, S43H, S43Q, K62E, K62R, L65Q, L65S, F71D, F71Y, F71E, F71Q,F71N, E82Q, E82N, E82K and F275H

As already indicated above, the variant may also contain conservativeamino acid substitutions.

The Non-Polypeptide Moiety

Based on the present disclosure the skilled person will be aware thatamino acid residues comprising other attachment groups may be introducedby substitution into the parent polypeptide, using the same approach asthat illustrated above with in vivo N-glycosylation sites. For instance,one or more amino acid residues comprising an acid group (glutamic acidor aspartic acid), tyrosine or lysine may be introduced into thepositions discussed above. In particular, one or more cysteine residuesmay be introduced in the positions discussed above.

As indicated further above the non-polypeptide moiety of the conjugatedvariant is preferably selected from the group consisting of a polymermolecule, a lipophilic compound, a sugar moiety (by way of in vivoglycosylation) and an organic derivatizing agent. All of these agentsmay confer desirable properties to the variant polypeptide, inparticular increased AUC_(iv), increased functional in vivo half-lifeand/or increased plasma half-life. The variant polypeptide is normallyconjugated to only one type of non-polypeptide moiety, but may also beconjugated to two or more different types of non-polypeptide moieties,e.g. to a polymer molecule and a sugar moiety, to a lipophilic group anda sugar moiety, to an organic derivatizing agent and a sugar moiety, toa lipophilic group and a polymer molecule, etc. The conjugation to twoor more different non-polypeptide moieties may be done simultaneous orsequentially.

Methods of Preparing a Conjugated Variant of the Invention

In the following sections “Conjugation to a lipophilic compound”,“Conjugation to a polymer molecule”, “Conjugation to a sugar moiety” and“Conjugation to an organic derivatizing agent” conjugation to specifictypes of non-polypeptide moieties is described. In general, a conjugatedvariant according to the invention may be produced by culturing anappropriate host cell under conditions conducive for the expression ofthe variant polypeptide, and recovering the variant polypeptide, whereina) the variant polypeptide comprises at least one N- or O-glycosylationsite and the host cell is an eukaryotic host cell capable of in vivoglycosylation, and/or b) the variant polypeptide is subjected toconjugation to a non-polypeptide moiety in vitro.

It will be understood that the conjugation should be designed so as toproduce the optimal molecule with respect to the number ofnon-polypeptide moieties attached, the size and form of such molecules(e.g. whether they are linear or branched), and the attachment site(s)in the polypeptide. The molecular weight of the non-polypeptide moietyto be used may, e.g., be chosen on the basis of the desired effect to beachieved. For instance, if the primary purpose of the conjugation is toachieve a conjugated variant having a high molecular weight (e.g. toreduce renal clearance) it is usually desirable to conjugate as few highmolecular weight non-polypeptide moieties as possible to obtain thedesired molecular weight. When a high degree of shielding is desirablethis may be obtained by use of a sufficiently high number of lowmolecular weight non-polypeptide moieties (e.g. with a molecular weightof from about 300 Da to about 5 kDa, such as a molecular weight of from300 Da to 2 kDa).

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the variant polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror hetero-polymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 500-20,000 Da, more preferably inthe range of about 500-15,000 Da, even more preferably in the range ofabout 2-12 kDa, such as in the range of about 3-10 kDa. When the term“about” is used herein in connection with a certain molecular weight,the word “about” indicates an approximate average molecular weight andreflects the fact that there will normally be a certain molecular weightdistribution in a given polymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer comprising different coupling groups, suchas a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-maleic acid anhydride, dextran, includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Another example of a polymer molecule is human albuminor another abundant plasma protein. Generally, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, non-antigenic,non-immunogenic, have various water solubility properties, and areeasily excreted from living organisms.

PEG is the preferred polymer molecule, since it has only few reactivegroups capable of cross-linking compared to, e.g., polysaccharides suchas dextran. In particular, mono-functional PEG, e.g. methoxypolyethyleneglycol (mPEG), is of interest since its coupling chemistry is relativelysimple (only one reactive group is available for conjugating withattachment groups on the polypeptide). Consequently, as the risk ofcross-linking is eliminated, the resulting conjugated variants are morehomogeneous and the reaction of the polymer molecules with the variantpolypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to the variantpolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA),succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC),N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), andtresylate (TRES)). Suitable activated polymer molecules are commerciallyavailable, e.g. from Shearwater Polymers, Inc., Huntsville, Ala., USA,or from PolyMASC Pharmaceuticals plc, UK.

Alternatively, the polymer molecules can be activated by conventionalmethods known in the art, e.g. as disclosed in WO 90/13540. Specificexamples of activated linear or branched polymer molecules for use inthe present invention are described in the Shearwater Polymers, Inc.1997 and 2000 Catalogs (Functionalized Biocompatible Polymers forResearch and pharmaceuticals, Polyethylene Glycol and Derivatives,incorporated herein by reference).

Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG,SG-PEG, and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branchedPEGs such as PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 andU.S. Pat. No. 5,643,575, both of which are incorporated herein byreference. Furthermore, the following publications, incorporated hereinby reference, disclose useful polymer molecules and/or PEGylationchemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No.5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673,EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503and EP 154 316.

Specific examples of activated PEG polymers particularly preferred forcoupling to cysteine residues, include the following linear PEGs:vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG);maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) andorthopyridyl-disulfide-PEG (OPSS-PEG), preferablyorthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEGpolymers will have a size of about 5 kDa, about 10 kD, about 12 kDa orabout 20 kDa.

The conjugation of the polypeptide variant and the activated polymermolecules is conducted by use of any conventional method, e.g. asdescribed in the following references (which also describe suitablemethods for activation of polymer molecules): Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.).

The skilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe variant polypeptide (examples of which are given further above), aswell as the functional groups of the polymer (e.g. being amine,hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide,vinysulfone or haloacetate). The PEGylation may be directed towardsconjugation to all available attachment groups on the variantpolypeptide (i.e. such attachment groups that are exposed at the surfaceof the polypeptide) or may be directed towards one or more specificattachment groups, e.g. the N-terminal amino group as described in U.S.Pat. No. 5,985,265 or to cysteine residues. Furthermore, the conjugationmay be achieved in one step or in a stepwise manner (e.g. as describedin WO 99/55377).

For PEGylation to cysteine residues (see above) the FVII or FVIIavariant is usually treated with a reducing agent, such as dithiothreitol(DDT) prior to PEGylation. The reducing agent is subsequently removed byany conventional method, such as by desalting. Conjugation of PEG to acysteine residue typically takes place in a suitable buffer at pH 6-9 attemperatures varying from 4° C. to 25° C. for periods up to 16 hours.

It will be understood that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form of such molecules (e.g. whether they arelinear or branched), and the attachment site(s) in the variantpolypeptide. The molecular weight of the polymer to be used may e.g. bechosen on the basis of the desired effect to be achieved.

In connection with conjugation to only a single attachment group on theprotein (e.g. the N-terminal amino group), it may be advantageous thatthe polymer molecule, which may be linear or branched, has a highmolecular weight, preferably about 10-25 kDa, such as about 15-25 kDa,e.g. about 20 kDa.

Normally, the polymer conjugation is performed under conditions aimed atreacting as many of the available polymer attachment groups with polymermolecules. This is achieved by means of a suitable molar excess of thepolymer relative to the polypeptide. Typically, the molar ratios ofactivated polymer molecules to polypeptide are up to about 1000-1, suchas up to about 200-1, or up to about 100-1. In some cases the ration maybe somewhat lower, however, such as up to about 50-1, 10-1, 5-1, 2-1 or1-1 in order to obtain optimal reaction.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person. A preferred example is cyanuric chloride(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat.No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed.,24, 375-378).

Subsequent to the conjugation, residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules are removed by a suitable method.

It will be understood that depending on the circumstances, e.g. theamino acid sequence of the variant polypeptide, the nature of theactivated PEG compound being used and the specific PEGylationconditions, including the molar ratio of PEG to polypeptide, varyingdegrees of PEGylation may be obtained, with a higher degree ofPEGylation generally being obtained with a higher ratio of PEG tovariant polypeptide. The PEGylated variant polypeptides resulting fromany given PEGylation process will, however, normally comprise astochastic distribution of conjugated polypeptide variants havingslightly different degrees of PEGylation.

Coupling to a Sugar Moiety

In order to achieve in vivo glycosylation of a FVII molecule comprisingone or more glycosylation sites the nucleotide sequence encoding thevariant polypeptide must be inserted in a glycosylating, eucaryoticexpression host. The expression host cell may be selected from fungal(filamentous fungal or yeast), insect or animal cells or from transgenicplant cells. In one embodiment the host cell is a mammalian cell, suchas a CHO cell, BHK or HEK, e.g. HEK 293, cell, or an insect cell, suchas an SF9 cell, or a yeast cell, e.g. S. cerevisiae or Pichia pastoris,or any of the host cells mentioned hereinafter.

Covalent in vitro coupling of sugar moieties (such as dextran) to aminoacid residues of the variant polypeptide may also be used, e.g. asdescribed, for example in WO 87/05330 and in Aplin etl al., CRC CritRev. Biochem, pp. 259-306,1981. The in vitro coupling of sugar moietiesor PEG to protein- and peptide-bound Gln-residues can be carried out bytransglutaminases (TGases). Transglutaminases catalyse the transfer ofdonor amine-groups to protein- and peptide-bound Gln-residues in aso-called cross-linking reaction. The donor-amine groups can be protein-or peptide-bound, such as the s-amino-group in Lys-residues or it can bepart of a small or large organic molecule. An example of a small organicmolecule functioning as amino-donor in TGase-catalysed cross-linking isputrescine (1,4-diaminobutane). An example of a larger organic moleculefunctioning as amino-donor in TGase-catalysed cross-linking is anamine-containing PEG (Sato et al., 1996, Biochemistry 35, 13072-13080).

TGases, in general, are highly specific enzymes, and not everyGln-residues exposed on the surface of a protein is accessible toTGase-catalysed cross-linking to amino-containing substances. On thecontrary, only few Gln-residues are naturally functioning as TGasesubstrates but the exact parameters governing which Gln-residues aregood TGase substrates remain unknown. Thus, in order to render a proteinsusceptible to TGase-catalysed cross-linking reactions it is often aprerequisite at convenient positions to add stretches of amino acidsequence known to function very well as TGase substrates. Several aminoacid sequences are known to be or to contain excellent natural TGasesubstrates e.g. substance P, elafin, fibrinogen, fibronectin, α₂-plasmininhibitor, α-caseins, and β-caseins.

Conjugation to an Organic Derivatizing Agent

Covalent modification of the variant polypeptide may be performed byreacting one or more attachment groups of the variant polypeptide withan organic derivatizing agent. Suitable derivatizing agents and methodsare well known in the art. For example, cysteinyl residues most commonlyare reacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction withdiethylpyrocarbonateat pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful. The reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0. Lysinyl and amino terminal residues are reacted with succinicor other carboxylic acid anhydrides. Derivatization with these agentshas the effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione and transaminase-catalyzed reactionwith glyoxylate. Arginyl residues are modified by reaction with one orseveral conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.

Furthermore, these reagents may react with the groups of lysine as wellas the arginine guanidino group. Carboxyl side groups (aspartyl orglutamyl) are selectively modified by reaction with carbodiimides(R—N═C═N—R′), where R and R′ are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Conjugation to a Lipophilic Compound

The variant polypeptide and the lipophilic compound may be conjugated toeach other, either directly or by use of a linker. The lipophiliccompound may be a natural compound such as a saturated or unsaturatedfatty acid, a fatty acid diketone, a terpene, a prostaglandin, avitamine, a carotenoide or steroide, or a synthetic compound such as acarbon acid, an alcohol, an amine and sulphonic acid with one or morealkyl-, aryl-, alkenyl- or other multiple unsaturated compounds. Theconjugation between the variant polypeptide and the lipophilic compound,optionally through a linker may be done according to methods known inthe art, e.g. as described by Bodanszky in Peptide Synthesis, JohnWiley, New York, 1976 and in WO 96/12505.

Methods of Preparing a Polypeptide Variant of the Invention

The polypeptide variant of the present invention may be produced by anysuitable method known in the art. Such methods include constructing anucleotide sequence encoding the polypeptide variant and expressing thesequence in a suitable transformed or transfected host. Preferably, thehost cell is a gammacarboxylating host cell such as a mammalian cell.However, polypeptide variants of the invention may be produced, albeitless efficiently, by chemical synthesis or a combination of chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

A nucleotide sequence encoding a polypeptide of the invention may beconstructed by isolating or synthesizing a nucleotide sequence encodingthe parent FVII, such as hFVII with the amino acid sequence shown in SEQID NO:2 and then changing the nucleotide sequence so as to effectintroduction (i.e. insertion or substitution) or removal (i.e. deletionor substitution) of the relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence is prepared by chemical synthesis, e.g. by using anoligonucleotide synthesizer, wherein oligonucleotides are designed basedon the amino acid sequence of the desired polypeptide, and preferablyselecting those codons that are favored in the host cell in which therecombinant polypeptide will be produced. For example, several smalloligonucleotides coding for portions of the desired polypeptide may besynthesized and assembled by PCR, ligation or ligation chain reaction(LCR) (Barany, PNAS 88:189-193, 1991). The individual oligonucleotidestypically contain 5′ or 3′ overhangs for complementary assembly.

Alternative nucleotide sequence modification methods are available forproducing polypeptide variants for high throughput screening, forinstance methods which involve homologous cross-over such as disclosedin U.S. Pat. No. 5,093,257, and methods which involve gene shuffling,i.e. recombination between two or more homologous nucleotide sequencesresulting in new nucleotide sequences having a number of nucleotidealterations when compared to the starting nucleotide sequences. Geneshuffling (also known as DNA shuffling) involves one or more cycles ofrandom fragmentation and reassembly of the nucleotide sequences,followed by screening to select nucleotide sequences encodingpolypeptides with desired properties. In order for homology-basednucleic acid shuffling to take place, the relevant parts of thenucleotide sequences are preferably at least 50% identical, such as atleast 60% identical, more preferably at least 70% identical, such as atleast 80% identical. The recombination can be performed in vitro or invivo.

Examples of suitable in vitro gene shuffling methods are disclosed byStemmer et al. (1994), Proc. Natl. Acad. Sci. USA; vol. 91, pp.10747-10751; Stemmer (1994), Nature, vol. 370, pp. 389-391; Smith(1994), Nature vol. 370, pp. 32⁴-325; Zhao et al., Nat. Biotechnol.1998, Mar; 16(3): 258-61; Zhao H. and Arnold, F B, Nucleic AcidsResearch, 1997, Vol. 25. No. 6 pp. 1307-1308; Shao et al., Nucleic AcidsResearch 1998, Jan 15; 26(2): pp. 681-83; and WO 95/17413.

An example of a suitable in vivo shuffling method is disclosed in WO97/07205. Other techniques for mutagenesis of nucleic acid sequences byin vitro or in vivo recombination are disclosed e.g. in WO 97/20078 andU.S. Pat. No. 5,837,458. Examples of specific shuffling techniquesinclude “family shuffling”, “synthetic shuffling” and “in silicoshuffling”.

Family shuffling involves subjecting a family of homologous genes fromdifferent species to one or more cycles of shuffling and subsequentscreening or selection. Family shuffling techniques are disclosed e.g.by Crameri et al. (1998), Nature, vol. 391, pp. 288-291; Christians etal. (1999), Nature Biotechnology, vol. 17, pp. 259-264; Chang et al.(1999), Nature Biotechnology, vol. 17, pp. 793-797; and Ness et al.(1999), Nature Biotechnology, vol. 17, 893-896.

Synthetic shuffling involves providing libraries of overlappingsynthetic oligonucleotides based e.g. on a sequence alignment ofhomologous genes of interest. The synthetically generatedoligonucleotides are recombined, and the resulting recombinant nucleicacid sequences are screened and if desired used for further shufflingcycles. Synthetic shuffling techniques are disclosed in WO 00/42561.

In silico shuffling refers to a DNA shuffling procedure, which isperformed or modelled using a computer system, thereby partly orentirely avoiding the need for physically manipulating nucleic acids.Techniques for in silico shuffling are disclosed in WO 00/42560.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide is insertedinto a recombinant vector and operably linked to control sequencesnecessary for expression of the FVII in the desired transformed hostcell.

It should of course be understood that not all vectors and expressioncontrol sequences function equally well to express the nucleotidesequence encoding the polypeptide variants described herein. Neitherwill all hosts function equally well with the same expression system.However, one of skill in the art may make a selection among thesevectors, expression control sequences and hosts without undueexperimentation. For example, in selecting a vector, the host must beconsidered because the vector must replicate in it or be able tointegrate into the chromosome. The vector's copy number, the ability tocontrol that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, should also beconsidered. In selecting an expression control sequence, a variety offactors should also be considered. These include, for example, therelative strength of the sequence, its controllability, and itscompatibility with the nucleotide sequence encoding the polypeptide,particularly as regards potential secondary structures. Hosts should beselected by consideration of their compatibility with the chosen vector,the toxicity of the product coded for by the nucleotide sequence, theirsecretion characteristics, their ability to fold the polypeptide variantcorrectly, their fermentation or culture requirements, and the ease ofpurification of the products coded for by the nucleotide sequence.

The recombinant vector may be an autonomously replicating vector, i.e. avector, which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector is one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector, in which the nucleotidesequence encoding the polypeptide variant of the invention is operablylinked to additional segments required for transcription of thenucleotide sequence. The vector is typically derived from plasmid orviral DNA. A number of suitable expression vectors for expression in thehost cells mentioned herein are commercially available or described inthe literature. Useful expression vectors for eukaryotic hosts, include,for example, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectorsare, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jola, Calif., USA). Useful expression vectorsfor yeast cells include the 2μ plasmid and derivatives thereof, the POT1vector (U.S. Pat. No. 4,931,373), the pJSO37 vector described in Okkels,Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C(Invitrogen). Useful vectors for insect cells include pVL941, pBG311(Cate et al., “Isolation of the Bovine and Human Genes for MullerianInhibiting Substance And Expression of the Human Gene In Animal Cells”,Cell, 45, pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both availablefrom Invitrogen). Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, includingpBR322, pET3a and pET12a (both from Novagen Inc., WI, USA), wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages.

Other vectors for use in this invention include those that allow thenucleotide sequence encoding the polypeptide variant to be amplified incopy number. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,“Construction Of A Modular Dihydrafolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The recombinant vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence (when the host cell is a mammalian cell) is the SV40 origin ofreplication. When the host cell is a yeast cell, suitable sequencesenabling the vector to replicate are the yeast plasmid 2μ replicationgenes REP 1-3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. ForSaccharomyces cerevisiae, selectable markers include ura3 and leu2. Forfilamentous fungi, selectable markers include amdS, pyrG, arcB, niaD andsC.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofthe polypeptide variant of the invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptidevariant. Such control sequences include, but are not limited to, aleader sequence, polyadenylation sequence, propeptide sequence,promoter, enhancer or upstream activating sequence, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter.

A wide variety of expression control sequences may be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription inmammalian cells include the early and late promoters of SV40 andadenovirus, e.g. the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus E1bregion polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol 1987 Aug. 20; 196(4):947-50).

In order to improve expression in mammalian cells a synthetic intron maybe inserted in the 5′ untranslated region of the nucleotide sequenceencoding the polypeptide. An example of a synthetic intron is thesynthetic intron from the plasmid pCI-Neo (available from PromegaCorporation, WI, USA).

Examples of suitable control sequences for directing transcription ininsect cells include the polyhedrin promoter, the P10 promoter, theAutographa californica polyhedrosis virus basic protein promoter, thebaculovirus immediate early gene 1 promoter and the baculovirus 39Kdelayed-early gene promoter, and the SV40 polyadenylation sequence.Examples of suitable control sequences for use in yeast host cellsinclude the promoters of the yeast α-mating system, the yeast triosephosphate isomerase (TPI) promoter, promoters from yeast glycolyticgenes or alcohol dehydrogenase genes, the ADH2-4c promoter, and theinducible GAL promoter. Examples of suitable control sequences for usein filamentous fungal host cells include the ADH3 promoter andterminator, a promoter derived from the genes encoding Aspergillusoryzae TAKA amylase triose phosphate isomerase or alkaline protease, anA. niger α-amylase, A. niger or A. nidulans glucoamylase, A. nidulansacetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1terminator and the ADH3 terminator. Examples of suitable controlsequences for use in bacterial host cells include promoters of the lacsystem, the trp system, the TAC or TRC system, and the major promoterregions of phage lambda.

The presence or absence of a signal peptide will, e.g., depend on theexpression host cell used for the production of the polypeptide variantto be expressed (whether it is an intracellular or extracellularpolypeptide) and whether it is desirable to obtain secretion. For use infilamentous fungi, the signal peptide may conveniently be derived from agene encoding an Aspergillus sp. amylase or glucoamylase, a geneencoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosalipase. The signal peptide is preferably derived from a gene encoding A.oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stableamylase, or A. niger glucoamylase. For use in insect cells, the signalpeptide may conveniently be derived from an insect gene (cf. WO90/05783), such as the Lepidopteran manduca sexta adipokinetic hormoneprecursor, (cf. U.S. Pat. No. 5,023,328), the honeybee melittin(Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al.,Protein Expression and Purification 4, 349-357 (1993) or humanpancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997).A preferred signal peptide for use in mammalian cells is that of hFVIIor the murine Ig kappa light chain signal peptide (Coloma, M (1992) J.Imm. Methods 152:89-104). For use in yeast cells suitable signalpeptides have been found to be the α-factor signal peptide from S.cereviciae (cf. U.S. Pat. No. 4,870,008), a modified carboxypeptidasesignal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), theyeast BAR1 signal peptide (cf. WO 87/02670), the yeast aspartic protease3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137), and the synthetic leader sequence TA57 (WO98/32867). For usein E. coli cells a suitable signal peptide have been found to be thesignal peptide ompA (EP581821).

The nucleotide sequence of the invention encoding a polypeptide variant,whether prepared by site-directed mutagenesis, synthesis, PCR or othermethods, may optionally include a nucleotide sequence that encode asignal peptide. The signal peptide is present when the polypeptidevariant is to be secreted from the cells in which it is expressed. Suchsignal peptide, if present, should be one recognized by the cell chosenfor expression of the polypeptide variant. The signal peptide may, e.g.be that normally associated with hFVII) or, alternatively, the signalpeptide may be from another source than hFVII, such as any of thosenormally associated with other human wild-type vitamin K-dependentpolypeptides. Furthermore, the signal peptide may be a signal peptidenormally expressed from the host cell or one which is not normallyexpressed from the host cell. Accordingly, the signal peptide may beprokaryotic, e.g. derived from a bacterium such as E. coli, oreukaryotic, e.g. derived from a mammalian, or insect or yeast cell.

Any suitable host may be used to produce the polypeptide variant,including bacteria (although not particularly preferred), fungi(including yeasts), plant, insect, mammal, or other appropriate animalcells or cell lines, as well as transgenic animals or plants. Examplesof bacterial host cells include grampositive bacteria such as strains ofBacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, orgramnegative bacteria, such as strains of E. coli. The introduction of avector into a bacterial host cell may, for instance, be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, MolecularGeneral Genetics 168: 111-115), using competent cells (see, e.g., Youngand Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221),electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journalof Bacteriology 169: 5771-5278). Examples of suitable filamentous fungalhost cells include strains of Aspergillus, e.g. A. oryzae, A. niger, orA. nidulans, Fusarium or Trichoderma. Fungal cells may be transformed bya process involving protoplast formation, transformation of theprotoplasts, and regeneration of the cell wall in a manner known per se.Suitable procedures for transformation of Aspergillus host cells aredescribed in EP 238 023 and U.S. Pat. No. 5,679,543. Suitable methodsfor transforming Fusarium species are described by Malardier et al.,1989, Gene 78: 147-156 and WO 96/00787. Examples of suitable yeast hostcells include strains of Saccharomyces, e.g. S. cerevisiae,Schizosaccharomyces, Klyveroinyces, Pichia, such as P. pastoris or P.methzanolica, Hansenula, such as H. Polymorpha or Yarrowia. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; Hinnen et al., 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1920: and as disclosed by ClontechLaboratories, Inc, Palo Alto, Calif., USA (in the product protocol forthe Yeastmaker™ Yeast Transformation System Kit). Examples of suitableinsect host cells include a Lepidoptora cell line, such as Spodopterafrugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (U.S.Pat. No. 5,077,214). Transformation of insect cells and production ofheterologous polypeptides therein may be performed as described byInvitrogen. Examples of suitable mammalian host cells include Chinesehamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkeycell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651));mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCCCRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCCCRL-1573)), as well as plant cells in tissue culture. Additionalsuitable cell lines are known in the art and available from publicdepositories such as the American Type Culture Collection, Rockville,Md. Also, the mammalian cell, such as a CHO cell, may be modified toexpress sialyltransferase, e.g. 1,6-sialyltransferase, e.g. as describedin U.S. Pat. No. 5,047,335, in order to provide improved glycosylationof the polypeptide variant.

In order to increase secretion it may be of particular interest toproduce the polypeptide variant of the invention together with anendoprotease, in particular a PACE (paired basic amino acid convertingenzyme) (e.g. as described in U.S. Pat. No. 5,986,079), such as a Kex2endoprotease (e.g. as described in WO 00/28065).

Methods for introducing exogeneous DNA into mammalian host cells includecalcium phosphate-mediated transfection, electroporation, DEAE-dextranmediated transfection, liposome-mediated transfection, viral vectors andthe transfection method described by Life Technologies Ltd, Paisley, UKusing Lipofectamin 2000. These methods are well known in the art ande.g. described by Ausbel et al. (eds.), 1996, Current Protocols inMolecular Biology, John Wiley & Sons, New York, USA. The cultivation ofmammalian cells are conducted according to established methods, e.g. asdisclosed in (Animal Cell Biotechnology, Methods and Protocols, Editedby Nigel Jenkins, 1999, Human Press Inc, Totowa, N.J., USA and HarrisonM A and Rae I F, General Techniques of Cell Culture, CambridgeUniversity Press 1997).

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide variant using methods known in the art. For example, thecell may be cultivated by shake flask cultivation, small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentersperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide variant is secreted into the nutrientmedium, the polypeptide can be recovered directly from the medium. Ifthe polypeptide variant is not secreted, it can be recovered from celllysates.

The resulting polypeptide variant may be recovered by methods known inthe art. For example, the polypeptide variant may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray drying, evaporation,or precipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation), HPLC, orextraction (see, e.g., Protein Purification, J.-C. Janson and LarsRyden, editors, VCH Publishers, New York, 1989).

Single chain polypeptide variants of the invention can be purified andactivated to two-chain polypeptide variants by a number of methods asdescribed in the literature (Broze and Majerus, 1980, J. Biol. Chem.255:1242-47 and Hedner and Kisiel, 1983, J. Clin. Invest. 71:1836-41).Another method whereby single chain polypeptide variant can be purifiedis by incorporation of Zn ions during purification as described in U.S.Pat. No. 5,700,914. In a preferred embodiment the polypeptide variant ispurified as a single chain polypeptide variant. The single chainpolypeptide variant is activated by either use of an immobilized enzyme(e.g. factors IIa, IXa, Xa and XIIa) or by autoactivation using apositively charged ion exchange matrix or the like.

It is advantageous to first purify the polypeptide variant in its singlechain form, then PEGylate (if desired) and last activate by one of themethods described above or by autoactivation as described by Pedersen etal, 1989, Biochemistry 28: 9331-36. The advantage of carrying outPEGylation before activation is that PEGylation of the new aminoterminalformed by cleavage of R152-I153 is avoided. PEGylation of this new aminoterminal would render the molecule inactive since the formation of ahydrogen bond between D242 and the amino terminal of I153 is necessaryfor activity.

Pharmaceutical Composition of the Invention and its Use

In a further aspect, the present invention relates to a composition, inparticular to a pharmaceutical composition, comprising a polypeptidevariant of the invention and a pharmaceutically acceptable carrier orexcipient.

The polypeptide variant or the pharmaceutical composition according tothe invention may be used as a medicament.

Due to the improved properties mentioned hereinbefore, the polypeptidevariants of the invention, or the pharmaceutical composition of theinvention, are particular useful for the treatment of uncontrollablebleeding events in trauma patients, thrombocytopenic patients, patientsin anticoagulant treatment, and cirrhosis patients with variceal bleeds,or other upper gastrointestinal bleedings, and in patients undergoingorthotopic liver transplantation, or liver resection (allowing fortransfusion free surgery).

Trauma is defined as an injury to living tissue caused by an extrinsicagent. It is the 4^(th) leading cause of death in the US and places alarge financial burden on the economy.

Trauma is classified as either blunt or penetrative. Blunt traumaresults in internal compression, organ damage and internal haemorrhagewhereas penetrative trauma (as the consequence of an agent penetratingthe body and destroying tissue, vessels and organs) results in externalhaemorrhage.

Haemorrhage, as a result of trauma, can start a cascade of problems. Forexample physiological compensation mechanisms are initiated with initialperipheral and mesenteric vasoconstriction to shunt blood to the centralcirculation. If circulation is not restored, hypovolemia shock (multipleorgan failure due to inadequate perfusion) ensues. Since tissuesthroughout the body become starved for oxygen, anaerobic metabolismbegins. However, the concomitant lactic acid leads the blood pH to dropand metabolic acidosis develops. If acidosis is severe and uncorrected,the patient may develop multisystem failure and die.

Although the majority of trauma patients are hypothermic on arrival inthe emergency room due to the environmental conditions at the scene,inadequate protection, intravenous fluid administration and ongoingblood loss worsen the hypothermic state. Deficiencies in coagulationfactors can result from blood loss or transfusions. Meanwhile, acidosisand hypothermia interfere with blood clotting mechanisms. Thuscoagulopathy develops, which in turn, may mask surgical bleeding sitesand hamper the control of mechanical bleeding.

Hypothermia, coagulopathy and acidosis are often characterised as the“trauma triad of death”

Trauma may be caused by several events. For example, road trafficaccidents result in many different types of trauma. Whilst some roadtraffic accidents are likely to result in penetrative trauma, many roadtraffic accidents are likely to inflict blunt trauma to both head andbody. However, these various types of trauma can all result incoagulopathy in the patient. Road traffic accidents are the leadingcause of accidental death in the US. There are over 42,000 deaths fromthem in the US each year. Many trauma patients die at the location ofthe accident either whilst being treated by the paramedics, before theyarrive or in transit to the ER.

Another example includes gunshot wounds. Gunshot wounds are traumas thatcan result in massive bleeding. They are penetrative and destroy tissueas the bullet passes through the body, whether it be in the torso or alimb. In the US about 40,000 people a year die from gunshot wounds

A further example includes falls. Falls result in a similar profile oftrauma type to road traffic accidents. By falling onto a solid object orthe ground from height can cause both penetrative and decelerative blunttrauma. In the US, falls are a common cause of accidental death,numbering about 13,000.

A still further example includes machinery accidents. A smaller numberof people die in the US from machinery accident related deaths, whetherstruck by, or entangled in machinery. The figures are small butsignificant—around 2,000.

A still further example includes stab wounds. Stab wounds arepenetrative injuries that can also cause massive bleeding. The organsmost likely to be damaged in a stab wound are the liver, small intestineand the colon.

Cirrhosis of the liver is the terminal sequel of prolonged repeatedinjury to the hepatic parenchyma. The end result is the formation ofbroad bands of fibrous tissue separating regenerative nodules that donot maintain the normal organization of liver lobules and thus causedeteriorated liver function. Patients have prolonged prothrombin timesas a result of the depletion of vitamin K-dependent coagulation factors.Pathogenetically, liver cirrhosis should be regarded as the final commonpathway of chronic liver injury, which can result from any form ofintense repeated prolonged liver cell injury. Cirrhosis of the liver maybe caused by direct liver injury, including chronic alcoholism, chronicviral hepatitis (types B, C, and D), and auto immune hepatitis as wellas by indirect injury by way of bile duct damage, including primarybiliary cirrhosis, primary sclerosing cholangitis and biliary atresia.Less common causes of cirrhosis include direct liver injury frominherited disease such as cystic fibrosis, alpha-1-antitrypsindeficiency, hemochromatosis, Wilson's disease, galactosernia, andglycogen storage disease.

Transplantation is primarily reserved for late stage cirrhotic patients,where it is the key intervention for treating the disease. To beeligible for transplantation, a patient must be classified as Child's Bor C, as well as meet additional criteria for selection. Last year, inthe US alone, 4,954 transplants were performed.

It has been estimated that there are 6,000 bleeding episodes associatedwith patients undergoing resection each year. This correlates with thereserved position of this procedure although seems slightly high incomparison with transplantation numbers.

Accurate data on the incidence of variceal bleeding is hard to obtain.The key facts known are that at the time of diagnosis, varices arepresent in about 60% of decompensated and 30% of compensated patientsand that about 30% of these patients with varices will experience ableed and that each episode of variceal bleeding is associated with a30% risk of mortality.

Thus, in a further aspect the present invention relates to a polypeptidevariant of the invention for the manufacture of a medicament for thetreatment of diseases or disorder wherein clot formation is desirable. Astill further aspect of the present invention relates to a method fortreating a mammal having a disease or disorder wherein clot formation isdesirable, comprising administering to a mammal in need thereof aneffective amount of the polypeptide variant or the pharmaceuticalcomposition of the invention.

Thrombocytopenia is caused by one of three mechanisms-decreased bonemarrow production, increased splenic sequestration, or accelerateddestruction of platelets. Thronmbocytopenia is a risk factor forhemorrhage, and platelet transfusion reduces the incidence of bleeding.The threshold for prophylactic platelet transfusion is 10,000/μl. Inpatients without fever or infections, a threshold of 5000/μl may besufficient to prevent spontaneous hemorrhage. For invasive procedures,50,000/μl platelets is the usual target level. In patients who developantibodies to platelets following repeated transfusions, bleeding can beextremely difficult to control.

Examples of diseases/disorders wherein increased clot formation isdesirable include, but is not limited to, hemorrhages, including brainhemorrhages, as well as patient with severe uncontrolled bleedings, suchas trauma. Further examples include patients undergoing livingtransplantations, patients undergoing resection and patients withvariceal bleedings.

The polypeptide variants of the invention is administered to patients ina therapeutically effective dose, normally one approximately parallelingthat employed in therapy with rFVII such as NovoSeven®, or at lowerdosage. By “therapeutically effective dose” herein is meant a dose thatis sufficient to produce the desired effects in relation to thecondition for which it is administered. The exact dose will depend onthe circumstances, and will be ascertainable by one skilled in the artusing known techniques. Normally, the dose should be capable ofpreventing or lessening the severity or spread of the condition orindication being treated. It will be apparent to those of skill in theart that an effective amount of a polypeptide variant or composition ofthe invention depends, inter alia, upon the disease, the dose, theadministration schedule, whether the polypeptide variant or compositionis administered alone or in conjunction with other therapeutic agents,the plasma half-life of the compositions, and the general health of thepatient. Preferably, the polypeptide variant or composition of theinvention is administered in an effective dose, in particular a dosewhich is sufficient to normalize the coagulation disorder.

The polypeptide variant of the invention is preferably administered in acomposition including a pharmaceutically acceptable carrier orexcipient. “Pharmaceutically acceptable” means a carrier or excipientthat does not cause any untoward effects in patients to whom it isadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company[1990]; Pharmaceutical Formulation Development of Peptides and Proteins,S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbookof Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]).

The polypeptide variant of the invention can be formulated intopharmaceutical compositions by well-known methods. Suitable formulationsare described by Remington's Pharmaceutical Sciences by E. W. Martin(Mark Publ. Co., 16th Ed., 1980).

The polypeptide variant of the invention can be used “as is” and/or in asalt form thereof. Suitable salts include, but are not limited to, saltswith alkali metals or alkaline earth metals, such as sodium, potassium,calcium and magnesium, as well as e.g. zinc salts. These salts orcomplexes may by present as a crystalline and/or amorphous structure.

The pharmaceutical composition of the invention may be administeredalone or in conjunction with other therapeutic agents. These agents maybe incorporated as part of the same pharmaceutical composition or may beadministered separately from the polypeptide variant of the invention,either concurrently or in accordance with another treatment schedule. Inaddition, the polypeptide variant or pharmaceutical composition of theinvention may be used as an adjuvant to other therapies.

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus, the methods are applicable to both humantherapy and veterinary applications. The pharmaceutical compositioncomprising the polypeptide variant of the invention may be formulated ina variety of forms, e.g. as a liquid, gel, lyophilized, or as acompressed solid. The preferred form will depend upon the particularindication being treated and will be apparent to one skilled in the art.

In particular, the pharmaceutical composition comprising the polypeptidevariant of the invention may be formulated in lyophilised or stablesoluble form. The polypeptide variant may be lyophilised by a variety ofprocedures known in the art. The polypeptide variant may be in a stablesoluble form by the removal or shielding of proteolytic degradationsites as described herein. The advantage of obtaining a stable solublepreparation lies in easier handling for the patient and, in the case ofemergencies, quicker action, which potentially can become life saving.The preferred form will depend upon the particular indication beingtreated and will be apparent to one of skill in the art.

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art,such as pumps or implantation. In some instances the formulations may bedirectly applied as a solution or spray.

Parentals

A preferred example of a pharmaceutical composition is a solutiondesigned for parenteral administration. Although in many casespharmaceutical solution formulations are provided in liquid form,appropriate for immediate use, such parenteral formulations may also beprovided in frozen or in lyophilized form. In the former case, thecomposition must be thawed prior to use. The latter form is often usedto enhance the stability of the active compound contained in thecomposition under a wider variety of storage conditions, as it isrecognized by those skilled in the art that lyophilized preparations aregenerally more stable than their liquid counterparts. Such lyophilizedpreparations are reconstituted prior to use by the addition of one ormore suitable pharmaceutically acceptable diluents such as sterile waterfor injection or sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide variant having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic surfactants or detergents, antioxidants and/orother miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse in the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Stabilizers refer to a broad category of excipients, which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilizing the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.). Additional miscellaneous excipientsinclude bulking agents or fillers (e.g. starch), chelating agents (e.g.EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) andcosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin micro spheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Sustained Release Preparations

Examples of sustained-release preparations include semi-permeablematrices of solid hydrophobic polymers containing the polypeptidevariant, the matrices having a suitable form such as a film ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization is depending on the mechanism involved. For example,if the aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The invention is further described in the following non-limitingexamples.

Materials and Methods

Accessible Surface Area (ASA)

The computer program Access (B. Lee and F. M. Richards, J. Mol. Biol.55: 379-400 (1971)) version 2 (© 1983 Yale University) is used tocompute the accessible surface area (ASA) of the individual atoms in thestructure. This method typically uses a probe-size of 1.4 Å and definesthe Accessible Surface Area (ASA) as the area formed by the center ofthe probe. Prior to this calculation all water molecules and allhydrogen atoms should be removed from the coordinate set, as shouldother atoms not directly related to the protein.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain with a valuerepresenting the ASA of the side chain atoms of that residue type in anextended Ala-x-Ala tripeptide (See Hubbard, Campbell & Thornton (1991)J. Mol. Biol. 220,507-530). For this example the CA atom is regarded asa part of the side chain of Glycine residues but not for the remainingresidues. The following table is used as standard 100% ASA for the sidechain: Ala  69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp 102.06 Å² Cys 96.69 Å² Gln 140.58 Å² Glu 134.61 Å² Gly  32.28 Å² His 147.00 Å² Ile137.91 Å² Leu 140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe 163.90 Å² Pro119.65 Å² Ser  78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr 176.61 Å² Val114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions. Thegamma-carboxy glutamic acids at positions 6, 7, 14, 16, 19, 20, 25, 26,29 and 35 are all defined as being 100% exposed.

Determining Distances Between Atoms

The distance between atoms is most easily determined using moleculargraphics software e.g. InsightII® v. 98.0, MSI INC.

Active Site Region

The active site region is defined as any residues having at least oneatom within 10 Å of any atom in the catalytic triad (residues H193,D242, S344).

Determination of Tissue Factor Binding Site

The TF binding site is defined as comprising of all residues havingtheir accessible surface area changed upon TF binding. This isdetermined by at least two ASA calculations; one on the isolatedligand(s) in the ligand(s)/receptor(s) complex and one on the completeligand(s)/receptor(s) complex.

Measurement of Reduced Sensitivity to Proteolytic Degradation

Proteolytic degradation can be measured using the assay described inU.S. Pat. No. 5,580,560, Example 5, where proteolysis isautoproteolysis.

Furthermore, reduced proteolysis can be tested in an in vivo model usingradiolabelled samples and comparing proteolysis of rhFVIIa and thepolypeptide variant of the invention by withdrawing blood samples andsubjecting these to SDS-PAGE and autoradiography.

Irrespectively of the assay used for determining proteolyticdegradation, “reduced proteolytic degradation” is intended to mean ameasurable reduction in cleavage compared to is that obtained by rhFVIIaas measured by gel scanning of Coomassie stained SDS-PAGE gels, HPLC oras measured by conserved catalytic activity in comparison to wild typeusing the tissue factor independent activity assay decribed below.

Determination of the Molecular Weight of Polypeptide Variants

The molecular weight of polypeptide variants is determined by eitherSDS-PAGE, get filtration, Western Blots, matrix assisted laserdesorption mass spectrometry or equilibrium centrifugation, e.g.SDS-PAGE according to Laemmli, U.K., Nature Vol 227 (1970), pp. 680-85.

TF-Independent Factor X Activation Assay

This assay has been described in detail on page 39826 in Nelsestuen etal., J Biol Chem, 2001; 276:39825-39831.

Briefly, the molecule to be assayed (either hFVIIa, rhFVIIa or thepolypeptide variant of the invention in its activated form) is mixedwith a source of phospholipid (phosphatidylcholine andphosphatidylserine in a ratio of 8:2 or phosphatidyleholine,phosphatidylserine and phosphatidylethanol in a ratio of 4:2:4) andFactor X in Tris buffer containing BSA. After a specified incubationtime the reaction is stopped by addition of excess EDTA. Theconcentration of factor Xa is then measured from absorbance change at405 nm after addition of a chromogenic substrate (S-2222, Chromogenix).After correction from background the tissue factor independent activityof rhFVIIa (a_(wt)) is determined as the absorbance change after 10minutes and the tissue factor independent activity of the polypeptidevariant of the invention (a_(variant)) is also determined as theabsorbance change after 10 minutes. The ratio between the activity ofthe polypeptide variant, in its activated form, and the activity ofrhFVIIa is defined as a_(variant)/a_(wt).

Clotting Assay

Clotting activity is measured in one-stage assays and clotting times arerecorded on a Thrombotrack IV coagulometer (MEDINOR). FVII depletedhuman plasma (American Diagnostica) is reconstituted and equilibrated atroom temperature for 15-20 minutes. 50 μl of plasma is then transferredto the coagulometer cups.

hFVIIa, rhFVIIa or variants are diluted in Glyoxaline Buffer (5.7 mMbarbiturate, 4.3 mM sodium citrate, 117 mM NaCl, 1 mg/mL BSA, pH 7.35).The samples are added to the cup in 50 μl and incubated at 37° C. for 2minutes.

Thromboplastin (MEDINOR) is reconstituted with water and CaCl₂ is added.The reaction is initiated by adding 0.1 ml thromboplastin containing 4.5mM CaCl₂.

Data are analysed using PRISM software.

TF-Independent Clotting Assay

This assay is performed as described above under “Clotting Assay” butwithout addition of thromboplastin.

Amidolytic Assay

The ability of the variants to cleave small peptide substrates can bemeasured using the chromogenic substrate S-2288(D-Ile-Pro-Arg-p-nitroanilide). The amidolytic activity may be measuredboth in the presence and absence of antithrombin III (ATIII).

HFVIIa, rhFVIIa or variant is diluted to 90 nM in assay buffer (50 mMNa-Hepes pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.1% BSA, 1U/ml Heparin).Furthermore, soluble TF (sTF) is diluted to 450 nM in assay buffer.ATIII is diluted to 900 nM in assay buffer. 120 μl of assay buffer ismixed with 20 μl of the FVIIa sample, 20 μl sTF and 20 μl ATIII or assaybuffer. The final concentrations of FVIIa, sTF and ATIII are 10, 50 and100 nM, respectively. After 5 min incubation at room temperature withgentle shaking, followed by 10 min incubation at 37° C., the reaction isstarted by addition of the S-2288 substrate to 1 mM and the absorptionat 405 nm is determined at several time points.

Thrombogram Assay

The effect of hFVIIa, rhFVIIa or variant on thrombin generation in humanplasma is tested in a modified version of the assay described on page589 in Hemker et al. in Thromb Haemost 2000; 83: 589-91. Briefly, themolecule to be assayed (either hFVIIa, rhFVIIa or variant) is mixed withnormal platelet rich plasma (PRP), normal platelet poor plasma (PPP) orFVII depleted PPP with or without the addition of recombinant humantissue factor (rTF), relipidated rTF or another source of TF (such asthromboplastin). A source of phospholipid (phosphatidylcholine andphosphatidylserine in a ratio of 8:2 or phosphatidylcholine,phosphatidylserine and phosphatidylethanol in a ratio of 4:2:4) can beadded.

The reaction is started by addition of a fluoregenic thrombin substrateand calcium chloride. The fluorescence is measured continuously and thethrombin amidolytic activity is calculated by calculating the slope ofthe fluorescence curve (the increase in fluorescence over time). In thisway the time until maximum thrombin amidolytic activity is obtained(T_(max)) and total thrombin work (area under the curve (AUC)) can becalculated.

The following procedure is used: PRP is obtained by centrifuging freshlydrawn blood at 250 g, 15° C. for 10 min. Blood coagulation is inhibitedeither by using citrate (13 mM tri-sodium citrate), corn trypsininhibitor (50-100 μg/ml blood) or a combination of citrate and corntrypsin inhibitor. The platelet count is adjusted to 3×10⁸/mL usingbuffer or autologous platelet poor plasma (PPP). PPP is obtained bydouble centrifugation of PRP at 1000 g, 15° C. for 10 min. FVIIdepletion is done by incubating PPP with a FVII specific monoclonalantibody coupled to a solid phase.

Per well of a 96-well microtiter plate 80 μl PRP is added and 20 μLbuffer containing rhFVII or variant to be tested in final concentrationsbetween 0.1 and 100 nM. rTF is added in 5 μL assay buffer to a finalconcentration of 1 pM. The assay buffer consists of 20 mM Hepes, 150 mMNaCl and 60 mg/ml BSA in distilled water. The reaction is started byadding 20 μL of the substrate solution containing 0.1 M calciumchloride. The assay plate and reagents are prewarmed to 37° C. and thereaction takes place at this temperature. The fluorimeter used is a BMGFluormeter with a excitation filter at 390 nm and an emission filter at460 nm. The fluorescence is measured in each well of 96-well clearbottom plates in 20-40 second interval over 30-180 minutes. Data areanalyzed using PRISM Software.

ELISA Assay

FVII/FVIIa (or variant) concentrations are determined by ELISA. Wells ofa microtiter late are coated with an antibody directed against theprotease domain using a solution of 2 μg/ml in PBS (100 μl per well).After 2 hours coating at R.T., the wells are washed 4 times with THTbuffer (100 mM NaCl, 50 mM Tris-HCl pH 7.2 0.05% Tween-20).Subsequently, 200 μl of 1% Casein (diluted from 2.5% stock using 100 mMNaCl, 50 mM Tris-HCl pH 7.2) is added per well for blocking. After 1 hrincubation at R.T., the wells are emptied, and 100 μl of sample(optionally diluted in dilution buffer (THT+0.1% Casein)) is added.After another incubation of 1 hr at room temperature, the wells arewashed 4 times with THT buffer, and 100 μl of a biotin-labelled antibodydirected against the EGF-like domain (1 μg/ml) is added. After another 1hr incubation at R.T., followed by 4 more washes with THT buffer, 100 μlof streptavidin-horse radish peroxidase (DAKO A/S, Glostrup, Denmark,1/10000 diluted) is added. After another 1 hr incubation at R.T.,followed by 4 more washes with THT buffer, 100 μl of TMB(3,3′,5,5′-tetramethylbenzidine, Kem-en-Tech A/S, Denmark) is added.After 30 min incubation at R.T. in the dark, 100 μl of 1 M H₂SO₄ isadded and OD_(450nm) is determined. A standard curve is prepared usingrhFVIIa (NovoSeven®).

Alternatively, FVII/FVIIa or variants may be quantified through the Gladomain rather than through the protease domain. In this ELISA set-up,wells are coated overnight with an antibody directed against theEGF-like domain and for detection, a calcium-dependent biotin-labelledmonoclonal anti-Gla domain antibody is used (2 μg/ml, 100 μl per well).In this set-up, 5 mM CaCl₂ is added to the THT and dilution buffers.

Whole Blood Assay

The whole blood clotting assay is performed as described by Elg et al.Thrombosis Res. 2001, 101(3):159-170.

Reconstituted Coagulation Assay

The reconstituted coagulation assay is performed as described by Van'tVeer et al. Blood 2000, 95(4), 1330-1335.

EXAMPLES Example 1

The X-ray structure of hFVIIa in complex with soluble tissue factor byBanner et. al., J Mol Biol, 1996; 285:2089 is used for this example. Itis noted that the numbering of residues in the reference does not followthe sequence. Here we have used the sequential numbering according toSEQ ID NO:2. The gamma-carboxy glutamic acids at positions 6, 7, 14, 16,19, 20, 25, 26, 29 and 35 are all here named GLU (three letterabbreviation) or E (one letter abbreviation). Residues 143-152 are notpresent in the structure.

Surface Exposure

Performing fractional ASA calculations on FVII fragments alone combinedwith the definition of accessibilities of non standard and/or missingresidues described in the methods resulted in the following residueshaving more than 25% of their side chain exposed to the surface: A1, N2,A3, F4, L5, E6, E7, L8, R9, P10, S12, L13, E14, E16, K18, E19, E20, Q21,S23, F24, E25, E26, R8, E29, F31, K32, D33, A34, E35, R36, K38, L39,W41, I42, S43, S45, G47, D48, Q49, A51, S52, S53, Q56, G58, S60, K62,D63, Q64, L65, Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82,T83, H84, K85, D86, D87, Q88, L89, I90, V92, N93, E94, G97, E99, S103,D104, H105, T106, G107, T108, K109, S111, R113, E116, G117, S119, L120,L121, A122, D123, G124, V125, S126, T128, P129, T130, V131, E132, 1140,L141, E142, K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152,G155, K157, V158, P160, K161, E163, L171, N173, G174, A175, N184, T185,1186, H193, K197, K199, N200, R202, N203, 1205, S214, E215, H216, D217,G218, D219, S222, R224, S232, T233, V235, P236, G237, T238, T239, N240,H249, Q250, P251, V253, T255, D256, E265, R266, T267, E270, R271, F275,V276, R277, F278, L280, L287, L288, D289, R290, G291, A292, T293, L295,E296, N301, M306, T307, Q308, D309, L311, Q312, Q313, R315, K316, V317,G318, D319, S320, P321, N322, T324, E325, Y326, Y332, S333, D334, S336,K337, K341, G342, H351, R353, G354, Q366, G367, T370, V371, G372, R379,E385, Q388, K389, R392, S393, E394, P395, R396, P397, G398, V399, L400,L401, R402, P404 and P406 (A1-S45 are located in the Gla domain, theremaining positions are located outside the Gla domain).

The following residues had more than 50% of their side chain exposed tothe surface: A1, A3, F4, L5, E6, E7, L8, R9, P10, E14, E16, K18, E19,E20, Q21, S23, E25, E26, E29, K32, A34, E35, R36, K38, L39, I42, S43,G47, D48, A51, S52, S53, Q56, G58, S60, K62, L65, Q66, S67, I69, F71,L73, P74, A75, E77, G78, R79, E82, H84, K85, D86, D87, Q88, L89, I90,V92, N93, E94, G97, T106, G107, T108, K109, S111, E116, S119, L121,A122, D123, G124, V131, E132, L141, E142, K143, R144, N145, A146, S147,K148, P149, Q150, G151, R152, G155, K157, P160, N173, G174, A175, K197,K199, N200, R202, S214, E215, H216, G218, R224, V235, P236, G237, T238,H249, Q250, V253, D256, T267, F275, R277, F278, L288, D289, R290, G291,A292, T293, L295, N301, M306, Q308, D309, L311, Q312, Q313, R315, K316,G318, D319, N322, E325, D334, K341, G354, G367, V371, E385, K389, R392,E394, R396, P397, G398, R402, P404 and P406 (A1-S43 are located in theGla domain, the remaining positions are located outside the Gla domain).

Tissue Factor Binding Site

Performing ASA calculations the following residues in human FVII changetheir ASA in the complex. These residues were defined as constitutingthe receptor binding site: L13, K18, F31, E35, R36, L39, F40, I42, S43,S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74, F76, E77, G78,R79, E82, K85, Q88, I90, V92, N93, E94, R271, A274, F275, V276, R277,F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325 and R379.

Active Site Region

The active site region is defined as any residue having at least oneatom within a distance of 10 Å from any atom in the catalytic triad(residues H193, D242, S344): I153, Q167, V168, L169, L170, L171, Q176,L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194,F195, D196, K197, 1198, W201, V228, I229, I230, P231, S232, T233, Y234,V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246,V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328,D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358,T359, G360, I361, V362, S363, W364, G365, C368, V376, Y377, T378, R379,V380, Q382, Y383, W386, L387, L400 and F405.

The Ridge of the Active Site Binding Cleft

The ridge of the active site binding cleft region was defined by visualinspection of the FVIIa structure 1FAK.pdb as: N173, A175, K199, N200,N203, D289, R290, G291, A292, P321 and T370.

Example 2

Design of an Expression Cassette for Expression of rhFVII in MammalianCells

The DNA sequence shown in SEQ ID NO:1, encompassing the short form ofthe full length cDNA encoding hFVII with its native short signal peptide(Hagen et al., 1986. PNAS 83:2412), was synthesized in order tofacilitate high expression in mammalian cells. First the ATG start codoncontext was modified according to the Kozak consensus sequence (Kozak,M. J Mol Biol 1987 Aug. 20; 196(4):947-50), so that there is a perfectmatch to the consensus sequence upstream of the ATG start codon.Secondly the open reading frame of the native cDNA was modified bymaking a bias in the codon usage towards the codons frequently used inhighly expressed human genes. Further, two translational stop codonswere inserted at the end of the open reading frame in order tofacilitate efficient translational stop. The fully synthetic andexpression optimized hFVII gene was assembled from 70-mer DNAoligonucleotides and finally amplified using end primers inserting BamHIand HindIII sites at the 5′ and 3′ ends respectively using standard PCRtechniques, which resulted in the following sequence:ggatcccgccaccatggtcagccaggccctccgcctcctgtgcctgctcctggggctgcagggctgcctggctgccgtcttcgtcacccaggaggaagcccatggcgtcctgcatcgccggcgccgggccaatgcctttctggaagagctccgccctggctccctggaacgcgaatgcaaagaggaacagtgcagctttgaggaagcccgggagattttcaaagacgctgagcggaccaaactgttttggattagctatagcgatggcgatcagtgcgcctccagcccttgccagaacgggggctcctgcaaagaccagctgcagagctatatctgcttctgcctgcctgcctttgaggggcgcaattgcgaaacccataaggatgaccagctgatttgcgtcaacgaaaacgggggctgcgagcagtactgcagcgatcacacgggcacgaagcggagctgccgctgccacgaaggctatagcctcctggctgacgggtgtcctgcacgcccacggtggaatacccttgcgggaagattcccattctagaaaagcggaacgctagcaaaccccagggccggatcgtcggcgggaaggtctgccctaagggggagtgcccctggcaggtcctgctcctggtcaacggggcccagctgtgcggcgggaccctcatcaataccatttgggtcgtgtccgccgctcactgcttcgataagattaagaattggcggaacctcatcgctgtgctcggcgaacacgatctgtccgagcatgacggggacgaacagtcccgccgggtggctcaggtcatcattccctccacctatgtgcctggcacgaccaatcacgatatcgctctgctccgcctccaccagcccgtcgtgctcaccgatcacgtcgtgcctctgttgcctgcctgagcggaccttagcgaacgcacgctggctttcgtccgctttagctctcgtgtccggctggggccagctgctcgaccggggcgctaccgctctcgagctgatggtgctcaacgtcccccggctgatgacccaggactgcctgcagcagtcccgcaaagtgggggactcccccaatatcacggagtatatgttttgcgctggctatagcgatggctccaaggatagctgcaagggggactccggcgggccccatgccacgcactatcgcgggacctggtacctcaccgggatcgtcagctggggccagggctgcgccaacggtggggcactttggcgtctacacgcgcgtcagccagtacattgagtggctgcagaagctcatgcggagcgaaccccggcccggggtgctcctgcgggcccctttcccttgat aaaagctt

A vector for the cloning of the generated PCR product encompassing theexpression cassette for hFVII was prepared by cloning the intron frompCINeo (Promega). The synthetic intron from pCI-Neo was amplified usingstandard PCR conditions and the primers: CBProFpr174:5′-AGCTGGCTAGCCACTGGGCAGGTAAGTATCA-3′ and CBProFpr175:5′-TGGCGGGATCCTTAAGAGCTGTAATTGAACT-3′resulting in a 332 bp PCR fragment. The fragment was cut with NheI andBamHI before cloning into pCDNA3.1/HygR (obtained from Invitrogen)resulting in PF#34.

The expression cassette for hFVII was cloned between the BamHI andHindIII sites of PF434, resulting in plasmid PF#226.

Example 3

Expression of Polypeptide Variants in CHO K1 Cells

The cell line CHO K1 (ATCC # CCL-61) is seeded at 50% confluence in T-25flasks using MEMα, 10% FCS (Gibco/BRL Cat # 10091), P/S and 5 μg/mlphylloquinone and allowed to grow until confluent. The confluent monocell layer is transfected with 5 μg of the relevant plasmid describedabove using the Lipofectamine 2000 transfection agent (Lifetechnologies) according to the manufacturer's instructions. Twenty fourhours post transfection a sample is drawn and quantified using e.g. anELISA recognizing the EGF1 domain of hFVII. At this time point relevantselection (e.g. Hygromycin B) may be applied to the cells with thepurpose of generating a pool of stable transfectants. When using CHO K1cells and the Hygromycin B resistance gene as selectable marker on theplasmid, this is usually achieved within one week.

Example 4

Generation of CHO K1 Cells Stably Expressing Polypeptide Variants.

A vial of CHO-K1 transfectant pool is thawed and the cells seeded in a175 cm² tissue flask containing 25 ml of MEMα, 10% FCS, phylloquinone (5μg/ml), 100 U/l penicillin, 100 μg/l streptomycin and grown for 24hours. The cells are harvested, diluted and plated in 96 well microtiterplates at a cell density of ½-1 cell/well. After a week of growth,colonies of 20-100 cells are present in the wells and those wellscontaining only one colony are labelled. After a further two weeks, themedia in all wells containing only one colony is substituted with 200 μlfresh medium. After 24 hours, a medium sample is withdrawn and analysedby e.g. ELISA. High producing clones are selected and used to produceFVII or variant on large scale.

Example 5

Purification of Polypeptide Variants and Subsequent Activation

FVII and FVII variants are purified as follows: The procedure isperformed at 4° C. The harvested culture media from large-scaleproduction is ultrafiltered using a Millipore TFF system with 30 KDacut-off Pellicon membranes. After concentration of the medium, citrateis added to 5 mM and the pH is adjusted to 8.6. If necessary, theconductivity is lowered to below 10 mS/cm. Subsequently, the sample isapplied to a Q-sepharose FF column, equilibrated with 50 mM NaCl, 10 mMTris pH 8.6. After washing the column with 100 mM NaCl, 10 mM Tris pH8.6, followed by 150 mM NaCl, 10 mM Tris pH 8.6, FVII is eluted using 10mM Tris, 25 mM NaCl, 35 mM CaCl₂, pH 8.6.

For the second chromatographic step, an affinity column is prepared bycoupling of a monoclonal Calcium-dependent antiGla-domain antibody toCNBr-activated Sepharose FF. About 5.5 mg antibody is coupled per mlresin. The column is equilibrated with 10 mM Tris, 100 mM NaCl, 35 mMCaCl₂, pH 7.5. NaCl is added to the sample to a concentration of 100 mMNaCl and the pH is adjusted to 7.4-7.6. After O/N application of thesample, the column is washed with 100 mM NaCl, 35 mM CaCl₂, 10 mM TrispH 7.5, and the FVII protein is eluted with 100 mM NaCl, 50 mM citrate,75 mM Tris pH 7.5.

For the third chromatographic, the conductivity of the sample is loweredto below 10 mS/cm, if necessary, and the pH is adjusted to 8.6. Thesample is then applied to a Q-sepharose column (equilibrated with 50 mMNaCl, 10 mM Tris pH 8.6) at a density around 3-5 mg protein per ml gelto obtain efficient activation. After application, the column is washedwith 50 mM NaCl, 10 mM Tris pH 8.6 for about 4 hours with a flow of 3-4column volumes (cv) per hour. The FVII protein is eluted using agradient of 0-100% of 500 mM NaCl, 10 mM Tris pH 8.6 over 40 cv. FVIIcontaining fractions are pooled.

For the final chromatographic step, the conductivity is lowered to below10 mS/cm. Subsequently, the sample is applied to a Q-sepharose column(equilibrated with 140 mM NaCl, 10 mM glycylglycine pH 8.6) at aconcentration of 3-5 mg protein per ml gel. The column is then washedwith 140 mM NaCl, 10 mM glycylglycine pH 8.6 and FVII is eluted with 140mM NaCl, 15 mM CaCl₂, 10 mM glycylglycine pH 8.6. The eluate is dilutedto 10 mM CaCl₂ and the pH is adjusted 6.8-7.2. Finally, Tween-80 isadded to 0.01% and the pH is adjusted to 5.5 for storage at −80° C.

1. A Factor VII (FVII) or Factor VIIa (FVIIa) polypeptide variant havingan amino acid sequence comprising 3-15 amino acid modifications relativeto human Factor VII (hFVII) or human Factor VIIa (hFVIIa) having theamino acid sequence shown in SEQ ID NO:2, wherein said amino acidsequence of the variant comprises an amino acid substitution in position10 and 32 and wherein a sugar moiety is covalently attached to anintroduced in vivo N-glycosylation site located outside the Gla domain.2. The variant according to claim 1, wherein said substitution inposition 10 is P10Q.
 3. The variant according to claim 1, wherein saidsubstitution in position 32 is K32E.
 4. The variant according to claim1, wherein said substitution in position 10 is P10Q and saidsubstitution in position 32 is K32E.
 5. The variant according to claim1, wherein said variant comprises at least one further amino acidmodification in the Gla domain. 6-15. (canceled)
 16. The variantaccording to claim 5, wherein said further modification in the Gladomain comprises an amino acid substitution in position
 34. 17. Thevariant according to claim 16, wherein a negatively charged amino acidresidue is introduced by substitution in position
 34. 18. The variantaccording to claim 17, wherein said substitution is A34E.
 19. Thevariant according to claim 18, wherein said variant comprises thesubstitutions P10Q+K32E+A34E. 20-25. (canceled)
 26. The variantaccording to claim 1, wherein said in vivo N-glycosylation site isintroduced by substitution. 27-28. (canceled)
 29. The variant accordingto claim 26, wherein said in vivo N-glycosylation site is introduced bya substitution selected from the group consisting of A51N, G58N, T106N,K109N, G124N, K143N+N145T, A175T, I205S, I205T, V253N, T267N,T267N+S269T, S314N+K316S, S314N+K316T, R315N+V317S, R315N+V317T,K316N+G318S, K316N+G318T, G318N, D334N and combinations thereof.
 30. Thevariant according to claim 29, wherein said in vivo N-glycosylation siteis introduced by a substitution selected from the group consisting ofA51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, I205T, V253N,T267N+S269T, S314N+K316T, R315N+V317T, K316N+G318T, G318N, D334N andcombinations thereof.
 31. The variant according to claim 30, whereinsaid in vivo N-glycosylation site is introduced by a substitutionselected from the group consisting of T106N, A175T, I205T, V253N,T267N+S269T and combinations thereof.
 32. The variant according to claim16, wherein one in vivo N-glycosylation site has been introduced bysubstitution.
 33. The variant according to claim 16, wherein two or morein vivo N-glycosylation sites have been introduced by substitution.34-40. (canceled)
 41. The variant according to claim 1, wherein saidvariant is in its activated form.
 42. The variant according to claim 1,wherein said variant, in its activated form, has at least 10% of theamidolytic activity of rhFVIIa when assayed in the “Amidolytic Assay”described herein.
 43. The variant according to claim 1, wherein saidvariant, in its activated form, has at least 10% of the clottingactivity of rhFVIIa when assayed in the “Clotting Assay” describedherein.
 44. A nucleotide sequence encoding the variant of claim
 1. 45.An expression vector comprising the nucleotide sequence of claim
 44. 46.A host cell comprising the nucleotide sequence of claim
 44. 47. The hostcell according to claim 46, wherein said host cell is agammacarboxylating cell capable of in vivo glycosylation.
 48. Apharmaceutical composition comprising the variant of claim 1, and apharmaceutical acceptable carrier or excipient. 49.-54. (canceled)
 55. Amethod for treating a mammal having a disease or a disorder wherein clotformation is desirable, comprising administering to a mammal in needthereof an effective amount of the pharmaceutical composition of claim48.
 56. The method according to claim 55, wherein said disease ordisorder is selected from the group consisting of hemorrhage, includingbrain hemorrhage, severe uncontrolled bleeding, such as trauma, bleedingin patients undergoing living transplantations, bleeding in patientsundergoing resection and variceal bleedings. 57-59. (canceled)